CLL1-binding nanobody, and antigen receptor and use thereof

Abstract

The present invention relates to the technical field of biomedicine, in particular to the field of cell therapy, specifically to a CLL1-binding nanobody or an antigen-binding fragment thereof, and further to a STAR comprising the CLL1-binding nanobody or the binding fragment thereof. The present invention further relates to a STAR binding to both CLL1 and LILRB4 targets, and uses of a STAR complex, cell and pharmaceutical composition comprising the STAR in the treatment and / or prevention of tumors.

Description

A nanobody bound to CLL1, an antigen receptor and its application Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a nanobody that specifically binds to CLL1 or its antigen-binding fragment, an antigen receptor, especially a dual-target STAR, and its application in the biomedical field. Background Technology

[0002] Acute myeloid leukemia (AML) is a malignant disease of myeloid hematopoietic stem / progenitor cells. It is characterized by abnormal proliferation of primitive and immature myeloid cells in the bone marrow and peripheral blood. Clinical manifestations include anemia, bleeding, infection and fever, organ infiltration, and metabolic abnormalities. Most cases are critically ill with a poor prognosis, and can be life-threatening if left untreated. Infants and young children are more susceptible to AML than adults, and it accounts for 30% of childhood leukemia cases. Current treatment methods include chemotherapy, supportive care, and hematopoietic stem cell transplantation.

[0003] The leukocyte immunoglobulin-like receptor (LILR) family plays a crucial role in the human immune system, including in the regulation and activation of immune cells. LILRB4, a member of this family, plays a vital role in immune cell inactivation. LILRB4 also serves as a specific biomarker in AML. Based on its specific expression in leukemia cells and myeloid immune cells, and its immunosuppressive function, LILRB4 has the potential to become an ideal therapeutic target for acute myelomonocytic and acute monocytic leukemia by eliminating tumor cells and improving the immunosuppressive environment. Deng M et al (Deng M, Gui L,Huang T,Zou Y,Chen B,Zhu H,Arase H,Xia N,Jiang Y,Collins R,You MJ,Homsi J,Unni N,Lewis C,Chen GQ,Fu YX,Liao XC,An Z,Zheng J,Zhang N,Zhang CC.LILRB4 signaling in leukaemia cells mediates T cell suppression and tumor A study in *Infiltration* (Nature, 2018, Oct; 562(7728):605-609) indicated that LILRB4, as an immunosuppressive receptor, coordinates the tumor invasion pathway in monocytic leukemia cells by creating an immunosuppressive microenvironment. Its expression level in monocytic AML cells is higher than in normal monocytic cells, and its expression level is negatively correlated with the survival rate of AML patients. Blocking LILRB4 signaling through knockout or antagonistic antibody methods can hinder the development of AML; therefore, LILRB4 is a potent target for the treatment of monocytic AML.Patent WO2024140710A1 describes a synthetic T-cell antigen receptor STAR and its application. This synthetic T-cell antigen receptor can specifically bind to LILRB4. The results show that the constructed STAR and STAR-T targeting LILRB4 can not only use LILRB4 as a tumor target antigen, but also use LILRB4 as a receptor in immunosuppressive cells, thereby killing tumor cells and reducing immunosuppression in tumor cells. It has greater potential for the treatment of relapsed and refractory AML FAB M4 / M5 type.

[0004] Another promising target for treating AML is CLL1. CLL1 (C-type lectin-like molecule 1) is not expressed in normal hematopoietic stem cells but is highly expressed in AML blast cells and leukemia stem cells. The most critical issue is that existing CLL1 antibodies lack affinity for the target and ligands exhibit off-target effects, necessitating the screening of new CLL1 antibody sequences with higher affinity.

[0005] On the other hand, while CAR-T and TCR-T cell therapies are rapidly developing for cancer treatment, STAR-T cells, derived from natural TCRs, lack the co-stimulatory signals required for T cell activation during activation, often affecting their proliferation and activation capabilities. Therefore, there is still a need in this field for improved STAR and corresponding STAR-T therapies. Summary of the Invention

[0006] To address the issues raised in existing technologies, this application screens nanobodies that specifically bind to CLL1. Furthermore, this application provides a synthetic T-cell receptor antigen receptor STAR and its application. This synthetic T-cell receptor antigen receptor can specifically bind to CLL1, and by targeting CLL1, TCRs and CARs can be modified to achieve better tumor treatment effects.

[0007] In addition, a synthetic T-cell receptor antigen receptor STAR and its application are provided. This synthetic T-cell receptor antigen receptor can specifically bind to two targets: CLL1 (C-type lectin-like molecule 1) and LILRB4 (leukocyte Ig-like receptor B4). While targeting CLL1, it can inhibit the tumor microenvironment by targeting LILRB4, thereby further improving the treatment of tumors, especially leukemia.

[0008] The specific plan is as follows:

[0009] A first aspect of the present invention provides a nanobody or antigen-binding fragment thereof that binds to CLL1, said nanobody or antigen-binding fragment comprising a heavy chain variable region, said heavy chain variable region comprising CDR1, CDR2 and CDR3, wherein:

[0010] CDR1 contains the amino acid sequence shown in SEQ ID NO:1, 2 or 3 or contains an amino acid sequence having three, two or one amino acid substitutions for the sequence shown in SEQ ID NO:1, 2 or 3.

[0011] CDR2 contains the amino acid sequence shown in SEQ ID NO: 4, 5, or 6, or contains an amino acid sequence having three, two, or one amino acid substitutions for the sequence shown in SEQ ID NO: 4, 5, or 6; and

[0012] CDR3 contains the amino acid sequence shown in SEQ ID NO: 7, 8 or 9 or contains an amino acid sequence having three, two or one amino acid substitutions for the sequence shown in SEQ ID NO: 7, 8 or 9.

[0013] In some specific embodiments, the nanobody or its antigen-binding fragment comprises CDR1, CDR2, and CDR3 as follows:

[0014] i) The CDR1 amino acid sequence comprises the amino acid sequence shown in SEQ ID NO 2 or contains an amino acid sequence having three, two, or one amino acid substitutions as shown in SEQ ID NO: 2; the CDR2 amino acid sequence comprises the amino acid sequence shown in SEQ ID NO: 5 or contains an amino acid sequence having three, two, or one amino acid substitutions as shown in SEQ ID NO: 5; and the CDR3 amino acid sequence comprises the amino acid sequence shown in SEQ ID NO: 8 or contains an amino acid sequence having three, two, or one amino acid substitutions as shown in SEQ ID NO: 8.

[0015] ii) The CDR1 amino acid sequence comprises or contains an amino acid sequence having three, two, or one amino acid substitutions as shown in SEQ ID NO: 1; the CDR2 amino acid sequence comprises or contains an amino acid sequence having three, two, or one amino acid substitutions as shown in SEQ ID NO: 4; and the CDR3 amino acid sequence comprises or contains an amino acid sequence having three, two, or one amino acid substitutions as shown in SEQ ID NO: 7; or

[0016] iii) The CDR1 amino acid sequence comprises the amino acid sequence shown in SEQ ID NO: 3 or contains an amino acid sequence having three, two, or one amino acid substitutions as shown in SEQ ID NO: 3; the CDR2 amino acid sequence comprises the amino acid sequence shown in SEQ ID NO: 6 or contains an amino acid sequence having three, two, or one amino acid substitutions as shown in SEQ ID NO: 6; and the CDR3 amino acid sequence comprises the amino acid sequence shown in SEQ ID NO: 9 or contains an amino acid sequence having three, two, or one amino acid substitutions as shown in SEQ ID NO: 9.

[0017] In some specific embodiments, the nanobody or its antigen-binding fragment further includes an FR region, preferably FR1-4 containing a heavy chain variable region.

[0018] Preferably, the FR1-4 are each independently derived from humans or non-human animals, such as mice.

[0019] The nanobody or its antigen-binding fragment includes FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 sequentially from the N-terminus to the C-terminus.

[0020] Preferably, the FR1 comprises any of the amino acid sequences described in SEQ ID NO: 16-18.

[0021] Preferably, the FR2 comprises any of the amino acid sequences described in SEQ ID NO: 19-20.

[0022] Preferably, the FR3 comprises any of the amino acid sequences described in SEQ ID NO: 21-23.

[0023] Preferably, the FR4 comprises the amino acid sequence described in SEQ ID NO: 24.

[0024] In some specific embodiments, the nanobody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 25, 26 or 27, or comprises an amino acid sequence having more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95% or more of homology with the amino acid sequence shown in SEQ ID NO: 25, 26 or 27.

[0025] A second aspect of the present invention provides a fusion protein comprising the nanobody or its antigen-binding fragment described in the first aspect.

[0026] The fusion protein also includes one or more of the following: protein tag, fluorescent protein, enzyme-catalyzed active protein, antibody Fc fragment, toxin protein, maltose-binding protein, or glutathione transferase.

[0027] The antibody Fc fragment can be an Fc fragment of an IgG antibody. The IgG antibody can be IgG1, IgG2, IgG3, or IgG4. Preferably, the sequence of the antibody Fc fragment is as shown in SEQ ID NO: 163.

[0028] Preferably, the protein tag includes a histidine His tag, a human influenza virus hemagglutinin HA tag, a FLAG tag, or a Myc tag.

[0029] Preferably, the fluorescent protein includes green fluorescent protein, blue fluorescent protein, or red fluorescent protein.

[0030] Preferably, the enzyme-catalyzing active protein includes alkaline phosphatase, horseradish peroxidase, or luminescent enzyme.

[0031] The antibody Fc fragment is linked to one or two nanobodies or their antigen-binding fragments.

[0032] The antibody Fc fragment is connected to the nanobody or its antigen-binding fragment via a linker, such as GS (SEQ ID NO: 86), GGS (SEQ ID NO: 76), or G4S (SEQ ID NO: 66).

[0033] Preferably, the two nanobodies or their antigen-binding fragments can be the same or different nanobodies or their antigen-binding fragments.

[0034] More preferably, the antibody Fc fragment is linked to NCLL02 and / or NCLL05.

[0035] In some specific embodiments, the antibody Fc fragment is linked to NCLL02 and other CLL1 antibodies (e.g., NCLL05 or NCLL06, the sequences of which are shown in Table 13).

[0036] In some specific embodiments, the antibody Fc fragment is linked to NCLL05 and other CLL1 antibodies (e.g., NCLL02 or NCLL06, the sequences of which are shown in Table 13).

[0037] In some specific embodiments, the antibody Fc fragment is linked to NCLL02 or NCLL05 and LILRB4 antibody or its antigen-binding fragment.

[0038] In one specific embodiment of the present invention, the sequence of the fusion protein comprising the antibody Fc fragment includes any one of SEQ ID NO: 164-166, or comprises an amino acid sequence having 70% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more homology with the amino acid sequences shown in SEQ ID NO: 164-166.

[0039] A third aspect of the present invention provides a STAR, said STAR comprising a first peptide chain and a second peptide chain.

[0040] The first peptide chain and / or the second peptide chain include the nanobody or its antigen-binding fragment described in the first aspect.

[0041] Preferably, the first peptide chain comprises a CLL1 single-chain antibody or nanobody, and the preferred CLL1 nanobody is selected from NCLL02, NCLL05 and / or NCLL06 (sequences are shown in Table 13).

[0042] Preferably, the second peptide chain comprises a CLL1 single-chain antibody or nanobody, and the preferred CLL1 nanobody is selected from NCLL02, NCLL05 and / or NCLL06 (sequences are shown in Table 13).

[0043] In some specific embodiments, the STAR includes a first peptide chain and a second peptide chain:

[0044] i) The first peptide chain includes a first target-binding region and a first constant region, and the second peptide chain includes a second target-binding region and a second constant region; or...

[0045] ii) The first peptide chain includes a first constant region, the second peptide chain includes a second constant region, and the first peptide chain or the second peptide chain includes a first target binding region;

[0046] Wherein the first target binding region and / or the second target binding region comprises one or more antigen binding regions, and the plurality of antigen binding regions may be the same or different or partially the same;

[0047] At least one of the antigen-binding regions in the first target-binding region and / or the second target-binding region contains a nanobody or antigen-binding fragment of the nanobody that specifically binds to CLL1 as described in the first aspect.

[0048] The antigen-binding regions in the first and / or second target-binding regions of the STAR further include antibodies or antigen-binding fragments that specifically bind to LILRB4, EGLN3, CD33, SIGLEC-6, CD7, CD123, CD47, CD70, CD55, CD117, or CD82.

[0049] Preferably, the antibody or its antigen-binding fragment is selected from scFv or nanobodies.

[0050] In some specific implementations, the STAR includes any one of the following groups:

[0051] a) The first peptide chain includes a first TCR constant region; the second peptide chain includes, from the N-terminus to the C-terminus, at least one nanobody or its antigen-binding fragment as described in any of the first aspects, and a second TCR constant region;

[0052] b) The first peptide chain comprises, from the N-terminus to the C-terminus, any of the nanobodies or antigen-binding fragments described in the first aspect, and a first TCR constant region; the second peptide chain comprises, from the N-terminus to the C-terminus, an antibody binding to LILRB4 and a second TCR constant region.

[0053] c) The first peptide chain includes a first TCR constant region; the second peptide chain includes, from the N-terminus to the C-terminus, any of the nanobodies or antigen-binding fragments described in the first aspect, an antibody that binds to LILRB4, and a second TCR constant region.

[0054] d) The first peptide chain includes a first TCR constant region; the second peptide chain includes, from the N-terminus to the C-terminus, an antibody binding to LILRB4, any of the nanobodies or their antigen-binding fragments described in the first aspect, and a second TCR constant region.

[0055] e) The first peptide chain comprises, from the N-terminus to the C-terminus, an antibody that binds to LILRB4, any of the nanobodies or their antigen-binding fragments described in the first aspect, and a first TCR constant region; the second peptide chain comprises, from the N-terminus to the C-terminus, any of the nanobodies or their antigen-binding fragments described in the first aspect, an antibody that binds to LILRB4, and a second TCR constant region; wherein the C-terminus of the first TCR constant region and / or the second TCR constant region is connected to at least one functional domain or is not connected to a functional domain;

[0056] f) The first peptide chain, from its N-terminus to its C-terminus, sequentially comprises any of the nanobodies or antigen-binding fragments described in the first aspect, an antibody binding to LILRB4, and a first TCR constant region; the second peptide chain, from its N-terminus to its C-terminus, sequentially comprises any of the nanobodies or antigen-binding fragments described in the first aspect, an antibody binding to LILRB4, and a second TCR constant region; wherein the C-terminus of the first TCR constant region and / or the second TCR constant region is connected to at least one functional domain, or is not connected to a functional domain; or

[0057] g) The first peptide chain comprises, from the N-terminus to the C-terminus, an antibody binding to LILRB4, any of the nanobodies or antigen-binding fragments described in the first aspect, and a first TCR constant region; the second peptide chain comprises, from the N-terminus to the C-terminus, an antibody binding to LILRB4, any of the nanobodies or antigen-binding fragments described in the first aspect, and a second TCR constant region; wherein the C-terminus of the first TCR constant region and / or the second TCR constant region is connected to at least one functional domain or is not connected to a functional domain.

[0058] The first TCR constant region of the first peptide chain is a constant region of the TCRα chain or a variant thereof, or a constant region of the TCRβ chain or a variant thereof; the second TCR constant region of the second peptide chain is a constant region of the TCRβ chain or a variant thereof, or a constant region of the TCRα chain or a variant thereof; the TCR constant regions of the first peptide chain and the second peptide chain are not simultaneously constant regions of the TCRα chain or a variant thereof, or are not simultaneously constant regions of the TCRβ chain or a variant thereof; or

[0059] The first TCR constant region of the first peptide chain is a constant region of the TCRγ chain or a variant thereof, or a constant region of the TCRδ chain or a variant thereof, and the second TCR constant region of the second peptide chain is a constant region of the TCRδ chain or a variant thereof, or a constant region of the TCRγ chain or a variant thereof; the TCR constant regions of the first peptide chain and the second peptide chain are not simultaneously constant regions of the TCRγ chain or a variant thereof, or are not simultaneously constant regions of the TCRδ chain or a variant thereof.

[0060] In some specific embodiments, the antibody binding to LILRB4 is a nanobody comprising CDR1 shown in SEQ ID NO: 10, CDR2 shown in SEQ ID NO: 11, and CDR3 shown in SEQ ID NO: 12;

[0061] Or including nanobodies of CDR1 shown in SEQ ID NO: 13, CDR2 shown in SEQ ID NO: 14, and CDR3 shown in SEQ ID NO: 15.

[0062] In one specific embodiment of the present invention, the sequence of the LILRB4-binding nanobody includes the amino acid sequence shown in SEQ ID NO: 121; or, it includes an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more identity with the amino acid sequence shown in SEQ ID NO: 121.

[0063] The sequence of the nanobody that binds to LILRB4 may include the amino acid sequence shown in SEQ ID NO: 120; or, may contain an amino acid sequence that has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more or more identity with the amino acid sequence shown in SEQ ID NO: 120.

[0064] Preferably, the constant region of the TCRα chain is the constant region of the human or mouse TCRα chain;

[0065] Preferably, the constant region of the TCRβ chain is the constant region of the human or mouse TCRβ chain;

[0066] Preferably, the constant region of the TCRγ chain is the constant region of the human or mouse TCRγ chain;

[0067] Preferably, the constant region of the TCRδ chain is the constant region of the human or mouse TCRδ chain.

[0068] In some specific embodiments, the STAR, wherein the constant region variant comprises one or more mutations from the group consisting of:

[0069] i) N-terminal modification; ii) cysteine ​​substitution; iii) hydrophobic amino acid substitution.

[0070] In some specific embodiments, the native intracellular regions of the first TCR constant region and / or the second TCR constant region are deleted and / or the constant regions are humanized or mouse-derived.

[0071] Preferably, the mutations are those shown in Tables 14-15.

[0072] The intracellular deletion of the constant region includes the deletion of amino acids 136-137 in the α chain constant region; and / or the deletion of amino acids 167-172 in the β chain constant region.

[0073] In some specific implementations, the STAR,

[0074] 1) The sequence of the constant region of the TCRα chain or a variant thereof includes any one of SEQ ID NO: 28, 30, 106-113; and / or, the sequence of the constant region of the TCRβ chain or a variant thereof includes any one of SEQ ID NO: 29, 31, 114-119; or

[0075] 2) The sequence of the constant region of the TCRγ chain or a variant thereof includes SEQ ID NO: 32 or 104; and / or, the sequence of the constant region of the TCRδ chain or a variant thereof includes SEQ ID NO: 33 or 105.

[0076] In some specific embodiments, the first peptide chain and / or the second peptide chain have at least one exogenous intracellular functional domain attached to their C-terminus.

[0077] Preferably, the exogenous intracellular functional domain is connected to the C-terminus of the constant region of the first peptide chain and / or the second peptide chain that is missing from the intracellular region.

[0078] Preferably, the functional domain includes one or more of the following: co-stimulatory molecular fragments, co-inhibitory molecular fragments, cytokine receptor fragments, or immune cell regulatory factor fragments.

[0079] Preferably, the co-stimulatory molecule includes one or more of 2B4, DAP10, DAP12, OX40, CD40, ICOS, CD28, 4-1BB, CD27 or NKp80; more preferably, the co-stimulatory molecule includes OX40.

[0080] More preferably, the intracellular domain of the OX40 includes the amino acid sequence shown in SEQ ID NO: 125.

[0081] More preferably, the intracellular domain of CD40 includes the amino acid sequence shown in SEQ ID NO: 126.

[0082] More preferably, the intracellular domain of the ICOS includes the amino acid sequence shown in SEQ ID NO: 127.

[0083] More preferably, the intracellular domain of CD28 includes the amino acid sequence shown in SEQ ID NO: 128.

[0084] More preferably, the intracellular domain of the 4-1BB comprises the amino acid sequence shown in SEQ ID NO: 129.

[0085] More preferably, the intracellular domain of CD27 includes the amino acid sequence shown in SEQ ID NO: 130.

[0086] Preferably, the co-inhibitory molecules include one or more of TIM3, PD1, CTLA4, or LAG3.

[0087] Preferably, the cytokine receptors include one or more of the following: interleukin receptors, interferon receptors, tumor necrosis factor superfamily receptors, colony-stimulating factor receptors, chemokine receptors, or growth factor receptors, such as one or more of IL-2β, IL-7α, or IL-21.

[0088] Preferably, the immune cell regulatory factor is a T cell regulatory factor, such as the NIK domain.

[0089] Preferably, the functional domain is a fusion protein of a cytokine receptor and a human STAT5 activation module, such as a fusion protein of IL-2β and a human STAT5 activation module or a fusion protein of IL-7α and a human STAT5 activation module.

[0090] Preferably, the amino acid sequence of the human STAT5 activation module is shown in SEQ ID NO: 124.

[0091] More preferably, the amino acid sequence of the IL-2β includes SEQ ID NO: 131.

[0092] More preferably, the amino acid sequence of the IL-7α includes SEQ ID NO: 132.

[0093] More preferably, the amino acid sequence of the IL-21 includes SEQ ID NO: 133.

[0094] More preferably, the amino acid sequence of the fusion protein in which IL-2β binds to the human STAT5 activation module includes SEQ ID NO: 134.

[0095] More preferably, the amino acid sequence of the fusion protein in which IL-7α binds to the human STAT5 activation module includes SEQ ID NO: 135.

[0096] In some specific implementations, the STAR includes:

[0097] A) The first peptide chain contains a first TCR constant region and at least one functional domain from the N-terminus to the C-terminus; the second peptide chain contains at least one nanobody or antigen-binding fragment of any of the first aspects and a second TCR constant region from the N-terminus to the C-terminus.

[0098] B) The first peptide chain comprises, from the N-terminus to the C-terminus, any of the nanobodies or antigen-binding fragments described in the first aspect, and a first TCR constant region; the second peptide chain comprises, from the N-terminus to the C-terminus, an antibody binding to LILRB4, a second TCR constant region, and at least one functional domain.

[0099] C) The first peptide chain contains, from N-terminus to C-terminus, a first TCR constant region and at least one functional domain; the second peptide chain contains, from N-terminus to C-terminus, any of the nanobodies or antigen-binding fragments described in the first aspect, an antibody that binds to LILRB4, and a second TCR constant region.

[0100] D) The first peptide chain contains, from the N-terminus to the C-terminus, a first TCR constant region and at least one functional domain; the second peptide chain contains, from the N-terminus to the C-terminus, an antibody that binds to LILRB4, any of the nanobodies or their antigen-binding fragments described in the first aspect, and a second TCR constant region.

[0101] E) The first peptide chain, from N-terminus to C-terminus, sequentially comprises any of the nanobodies or antigen-binding fragments described in the first aspect, a first TCR constant region, and at least one functional domain; the second peptide chain, from N-terminus to C-terminus, sequentially comprises an antibody binding to LILRB4, a second TCR constant region, and at least one functional domain; F) The first peptide chain, from N-terminus to C-terminus, sequentially comprises any of the nanobodies or antigen-binding fragments described in the first aspect, a first TCR constant region, and at least one functional domain; the second peptide chain, from N-terminus to C-terminus, sequentially comprises an antibody binding to LILRB4 and a second TCR constant region;

[0102] G) The first peptide chain contains a first TCR constant region and at least one functional domain from the N-terminus to the C-terminus; the second peptide chain contains at least one nanobody or antigen-binding fragment of any of the first aspects, a second TCR constant region, and at least one functional domain from the N-terminus to the C-terminus.

[0103] H) The first peptide chain contains a first TCR constant region from the N-terminus to the C-terminus; the second peptide chain contains an antibody binding to LILRB4, any of the nanobodies or antigen-binding fragments described in the first aspect, a second TCR constant region, and at least one functional domain from the N-terminus to the C-terminus.

[0104] I) The first peptide chain contains a first TCR constant region; the second peptide chain contains, from the N-terminus to the C-terminus, any of the nanobodies or antigen-binding fragments described in the first aspect, an antibody that binds to LILRB4, a second TCR constant region, and at least one functional domain.

[0105] In some specific embodiments, the STAR, wherein the domains of the STAR (e.g., between functional domains and the C-terminus of the constant region, between functional domains tandemly on the same peptide chain, between antigen-binding regions and constant regions, or between antigen-binding regions tandemly on the same peptide chain) are directly connected by peptide bonds or by adapters.

[0106] Preferably, the connector is a (G4S)n connector or a (EAAAK)n connector, where n represents an integer from 1 to 10, for example, n represents 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0107] Preferably, the connector includes a rigid connector, a flexible connector, a pyrolytic connector, or a meaningless amino acid;

[0108] Preferably, the amino acid sequence of the rigid connector includes one or more of SEQ ID NO: 34-44; the amino acid sequence of the flexible connector includes one or more of SEQ ID NO: 45-97 or 103; and the cleavable connector includes one or more of SEQ ID NO: 98-102.

[0109] In some specific embodiments, the STAR, wherein the first peptide chain and the second peptide chain in the STAR are connected or not connected by a cleavable linker; preferably, the cleavable linker is a Furin-P2A linker, preferably, the Furin-P2A linker is as shown in the amino acid sequence of SEQ ID NO: 137.

[0110] In some specific embodiments, the STAR, wherein the first peptide chain is selected from SEQ ID NO: 143, 145, 147, 149, 151, 153, 155, 157, 159, 161 or 167; and / or

[0111] The amino acid sequence of the second peptide chain is selected from SEQ ID NO: 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 168, 169 or 170.

[0112] In one specific embodiment of the present invention, the first peptide chain is SEQ ID NO: 167, and the second peptide chain is SEQ ID NO: 168, SEQ ID NO: 169, or SEQ ID NO: 170. Alternatively, the first peptide chain is SEQ ID NO: 143, and the second peptide chain is SEQ ID NO: 144. Alternatively, the first peptide chain is SEQ ID NO: 145, and the second peptide chain is SEQ ID NO: 146. Alternatively, the first peptide chain is SEQ ID NO: 151, and the second peptide chain is SEQ ID NO: 152. Alternatively, the first peptide chain is SEQ ID NO: 153, and the second peptide chain is SEQ ID NO: 154. Alternatively, the first peptide chain is SEQ ID NO: 155, and the second peptide chain is SEQ ID NO: 156. Alternatively, the first peptide chain is SEQ ID NO: 157, and the second peptide chain is SEQ ID NO: 158. Alternatively, the first peptide chain is SEQ ID NO: 159, and the second peptide chain is SEQ ID NO: 160. Alternatively, the first peptide chain is SEQ ID NO: 161, and the second peptide chain is SEQ ID NO: 162. Alternatively, the first peptide chain is SEQ ID NO: 147, and the second peptide chain is SEQ ID NO: 148. Alternatively, the first peptide chain is SEQ ID NO: 149, and the second peptide chain is SEQ ID NO: 150.

[0113] Preferably, the functional domains contained in the first peptide chain may be the same as, different from, or partially the same as the functional domains contained in the second peptide chain.

[0114] In some embodiments, the STAR includes:

[0115] (A) The α chain and / or β chain have at least one functional domain connected to their C-terminus, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains; or,

[0116] (B) The γ chain and / or δ chain are connected to at least one functional structural domain at their C-terminus, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional structural domains.

[0117] In one specific embodiment of the present invention, the functional structural domain is OX40.

[0118] In a fourth aspect, the present invention provides a STAR complex comprising the STAR described in the third aspect, and one or more of CD3γ, CD3δ, CD3ε and CD3ζ.

[0119] Preferably, the amino acid sequence of CD3γ includes SEQ ID NO: 138.

[0120] Preferably, the amino acid sequence of CD3δ includes SEQ ID NO: 139.

[0121] Preferably, the amino acid sequence of CD3ε includes SEQ ID NO: 140.

[0122] Preferably, the amino acid sequence of CD3ζ includes SEQ ID NO: 136.

[0123] In a fifth aspect, the present invention provides an antigen receptor comprising the CLL1-binding nanobody or antigen-binding fragment thereof described in the first aspect, or the STAR described in the third aspect.

[0124] The antigen receptor is selected from TCR, STAR, CAR, or antibodies containing a constant region or an Fc region.

[0125] The Fc region can be the Fc region of an IgG antibody. The IgG antibody can be IgG1, IgG2, IgG3, or IgG4. Preferably, the sequence of the Fc region is as shown in SEQ ID NO: 163.

[0126] The antibody containing the Fc region comprises an Fc region linked to at least one nanobody or its antigen-binding fragment. The linking can be direct via peptide bonds or via a adapter. Preferably, the Fc region is linked to the nanobody or its antigen-binding fragment via a adapter, such as GS (SEQ ID NO: 86), GGS (SEQ ID NO: 76), or G4S (SEQ ID NO: 66).

[0127] In one specific embodiment of the present invention, the sequence of the antibody containing the Fc region includes any one of SEQ ID NO: 164-166, or contains an amino acid sequence having more than 70%, more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or more than 99.9% homology with the amino acid sequences shown in SEQ ID NO: 164-166.

[0128] In a sixth aspect, the present invention provides a nucleic acid that encodes the nanobody or its antigen-binding fragment described in the first aspect, the fusion protein described in the second aspect, the STAR described in the third aspect, the STAR complex described in the fourth aspect, or the antigen receptor described in the fifth aspect.

[0129] A seventh aspect of the present invention provides a carrier that contains the nucleic acid described in the sixth aspect.

[0130] Preferably, the vector expressing STAR comprises, from the 5' end to the 3' end, the following:

[0131] The nucleotide sequence encoding the first peptide chain, the nucleotide sequence encoding the linker peptide, and the nucleotide sequence encoding the second peptide chain; or,

[0132] The nucleotide sequence encoding the second peptide chain, the nucleotide sequence encoding the linker peptide, and the nucleotide sequence encoding the first peptide chain.

[0133] Preferably, the linker peptide is a cleavable linker, including but not limited to 2A peptides, such as Furin-P2A. Preferably, the amino acid sequence of Furin-P2A includes SEQ ID NO: 137.

[0134] Preferably, the vector is a eukaryotic vector or a prokaryotic vector, more preferably a plasmid or a viral vector, and even more preferably a lentiviral vector.

[0135] Preferably, the nucleic acid is operatively linked to a controllable sequence, wherein the controllable sequence includes one or more of the following: promoter, terminator, leader sequence, enhancer, transposon, intron, polyadenylated sequence, origin of replication, or multiple cloning site; preferably, the controllable sequence includes one or more of the following: hEF1a-HTLV promoter, WPRE element, LTR, SV40 polyA sequence, SV40 origin, or multiple cloning site.

[0136] In an eighth aspect, the present invention provides a host cell comprising the nucleic acid described in the sixth aspect or the vector described in the seventh aspect.

[0137] The ninth aspect of the present invention provides an immune cell that expresses the nanobody or its antigen-binding fragment described in the first aspect, the fusion protein described in the second aspect, the STAR described in the third aspect, the STAR complex described in the fourth aspect, or the antigen receptor described in the fifth aspect.

[0138] The immune cells mentioned include one or more of the following: lymphocytes, dendritic cells, peripheral blood mononuclear cells, macrophages, granulocytes, or mast cells.

[0139] Preferably, the lymphocytes are selected from T cells, TIL cells, NK cells, NKT cells, or B cells.

[0140] More preferably, the T cells include one or more of the following: memory stem cell-like T cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef), non-infiltrating regulatory T cells (Tregs), effector memory T cells (Tem), γδ T cells, or αβ T cells.

[0141] In a tenth aspect of the present invention, a method for preparing the immune cells described in the ninth aspect is provided, wherein the method comprises transfecting the nucleic acid described in the sixth aspect into immune cells for expression.

[0142] In an eleventh aspect of the present invention, the use of the nanobody or antigen-binding fragment thereof described in the first aspect, the fusion protein described in the second aspect, the STAR described in the third aspect, the STAR complex described in the fourth aspect, the antigen receptor described in the fifth aspect, the nucleic acid described in the sixth aspect, the carrier described in the seventh aspect, the host cell described in the eighth aspect, or the immune cell described in the ninth aspect in the preparation of products for the diagnosis, prevention, and / or treatment of tumors is provided.

[0143] The tumor in question is one that expresses, abnormally expresses, or overexpresses CLL1 or its mutants.

[0144] The tumor in question is one in which reducing CLL1 expression is beneficial for treatment. Preferably, the tumor in question is one in which reducing CLL1 and / or LILRB4 expression is beneficial for treatment.

[0145] The tumors mentioned include lymphoma, non-small cell lung cancer, leukemia, ovarian cancer, nasopharyngeal carcinoma, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma; wherein, the leukemia mentioned is selected from acute lymphoblastic leukemia, acute myeloid leukemia, myeloid leukemia, and chronic lymphocytic leukemia. Lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myeloid leukemia; the lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenström macroglobulinemia; the sarcoma is selected from osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma.

[0146] Preferably, the tumor is leukemia or lymphoma. Preferably, the leukemia is selected from acute lymphoblastic leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myeloid leukemia.

[0147] In one specific embodiment of the present invention, the tumor is leukemia (e.g., acute myeloid leukemia).

[0148] A twelfth aspect of the present invention provides a pharmaceutical composition comprising the nanobody or antigen-binding fragment thereof described in the first aspect, the fusion protein described in the second aspect, the STAR described in the third aspect, the STAR complex described in the fourth aspect, the antigen receptor described in the fifth aspect, the nucleic acid described in the sixth aspect, the carrier described in the seventh aspect, the host cell described in the eighth aspect, or the immune cell described in the ninth aspect. Preferably, it further comprises pharmaceutically acceptable excipients.

[0149] Preferably, the pharmaceutically acceptable excipients include one or more of the following: excipients, diluents, binders, lubricants, humectants, emulsifiers, preservatives, antioxidants, buffers, antibacterial agents, disintegrants, surfactants, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners, and flavorings.

[0150] The pharmaceutical composition described herein can be used to treat humans or non-human animals, such as non-human mammals.

[0151] The pharmaceutical composition can be administered via any suitable route of administration, such as gastrointestinal (e.g., oral) or non-gastrointestinal (e.g., intravenous, intramuscular, subcutaneous, intradermal, intra-organ, intranasal, intraocular, intravenous, intracerebral, intrathecal, transdermal, rectal, etc.).

[0152] The pharmaceutical composition may be any suitable dosage form, such as a gastrointestinal or non-gastrointestinal dosage form, preferably including but not limited to tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, microemulsions, suspensions, injections, sprays, aerosols, powder inhalers, lotions, ointments, plasters, pastes, patches, eye drops, nasal drops, sublingual tablets, suppositories, aerosols, effervescent tablets, pellets, gels, etc.

[0153] Various dosage forms of the pharmaceutical composition can be prepared according to conventional pharmaceutical manufacturing methods.

[0154] The pharmaceutical composition may contain 0.01-99.5% (specifically, 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%) of nanobodies or their antigen-binding fragments, fusion proteins, antigen receptors, STARs, STAR complexes, nucleic acids, carriers, host cells, and immune cells (especially STAR-T cells) in a weight ratio of 1%.

[0155] The pharmaceutical composition described herein can treat tumors.

[0156] In a thirteenth aspect, the present invention provides a diagnostic kit comprising the nanobody or antigen-binding fragment thereof described in the first aspect, the fusion protein described in the second aspect, the STAR described in the third aspect, the STAR complex described in the fourth aspect, the antigen receptor described in the fifth aspect, the nucleic acid described in the sixth aspect, the vector described in the seventh aspect, the host cell described in the eighth aspect, or the immune cell described in the ninth aspect.

[0157] The diagnostic kit described can diagnose whether a person has a tumor.

[0158] The fourteenth aspect of the present invention provides a method for preventing and / or treating tumors, the method comprising applying to an individual in need an effective amount of the nanobody or antigen-binding fragment thereof described in the first aspect, the fusion protein described in the second aspect, the STAR described in the third aspect, the STAR complex described in the fourth aspect, the antigen receptor described in the fifth aspect, the nucleic acid described in the sixth aspect, the carrier described in the seventh aspect, the host cell described in the eighth aspect, or the immune cell described in the ninth aspect, or the pharmaceutical composition described in the twelfth aspect.

[0159] The administration route may be gastrointestinal (e.g., oral) or non-gastrointestinal (e.g., intravenous, intramuscular, subcutaneous, intradermal, intra-organ, intranasal, intraocular, intravenous, intracerebral, intrathecal, transdermal, rectal, etc.), with intravenous injection being preferred.

[0160] The application is performed 1-10 times, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times, preferably 1-3 times.

[0161] The individual is a person, and the single dose is 10. 4 / kg-10 9 / kg, preferably 5×10 5 / kg-5×10 7 / kg.

[0162] The tumor in question is one that expresses, abnormally expresses, or overexpresses CLL1 or its mutants.

[0163] Preferably, the tumor is a tumor in which reducing CLL1 and / or LILRB4 expression is beneficial for treatment.

[0164] The tumors include lymphoma, non-small cell lung cancer, leukemia, ovarian cancer, nasopharyngeal carcinoma, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma; the lymphomas are selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenström macroglobulinemia; the sarcomas are selected from osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma.

[0165] Preferably, the tumor is leukemia or lymphoma.

[0166] Preferably, the leukemia is selected from acute lymphoblastic leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myeloid leukemia.

[0167] The "antigen-binding fragment" described in this invention is a portion of an antibody that retains the specific binding activity of the antibody; that is, any part of the antibody can specifically bind to an epitope on the antibody's target molecule. It includes, for example, Fab, Fab', F(ab')2, Fv, Fd, Fab'-SH, and variants of these fragments. For example, the heavy and / or light chains of the antibody, the variable regions of the heavy and / or light chains of the antibody, or one or more CDRs (Complementarity Determining Regions) from the heavy or light chains of the antibody. Specifically, Fab is a monovalent fragment consisting of VL, VH, CL, and CH1 domains. F(ab')2 is a divalent fragment comprising two Fab fragments connected by disulfide bonds in a hinge region. Fd is an Fd fragment consisting of VH and CH1 domains. Fv is an Fv fragment consisting of the VL and VH domains of a single arm of the antibody. Fab' is a Fab fragment having one or more cysteine ​​residues at the C-terminus of the CH1 domain. Fab'-SH is a Fab' whose constant domain contains cysteine ​​residues with at least one free thiol group. Here, VH represents the heavy chain variable region, VL represents the light chain variable region, and CL represents the light chain.

[0168] The “CDR” as used in this invention refers to the complementarity-determining region within the variable sequence of an antibody. For each variable region, there are three CDRs in each variable region of the heavy and light chains, referred to as CDR1, CDR2, and CDR3. The exact boundaries of these CDRs are defined differently depending on the system. The system described by Kabat et al. (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides a definitive residue numbering system applicable to antibody variable regions but also provides residue boundaries defining the three CDRs. These CDRs can be referred to as Kabat CDRs. Each complementarity-determining region may contain amino acid residues from what is defined as a “complementarity-determining region” as by Kabat. Chothia et al. (Chothia & Lesk, J. Mol. Biol, 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (-1989)) found that Kabat… Certain sub-regions within a CDR adopt nearly identical peptide skeleton imagery, despite significant diversity at the amino acid sequence level. These sub-regions are referred to as L1, L2, and L3, or H1, H2, and H3, where "L" and "H" represent the light and heavy chain regions, respectively. These regions may be called Chothia CDRs, with boundaries overlapping with Kabat CDRs. Other CDR boundary definitions may not strictly adhere to one of the aforementioned systems but will still overlap with Kabat CDRs; the methods used herein can utilize CDRs defined according to any of these systems, although the preferred embodiment uses CDRs defined by Kabat or Chothia. The term "antibody variable region" refers to the portion of the amino acid sequence in the light and heavy chains of the antibody molecule that includes the complementarity-determining regions (CDRs, i.e., CDR1, CDR2, and CDR3) and the framework region (French, FR). VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain.

[0169] "Co-stimulatory molecular fragments" refer to fragments in STAR that can function as co-stimulatory molecules, including full-length or partial fragments with co-stimulatory molecular functions.

[0170] "Co-inhibitory molecular fragments" refer to fragments that can function as co-inhibitory molecules in STAR, including full-length or partial fragments with co-inhibitory molecular functions.

[0171] "Cytokine receptor fragments" refer to fragments in STAR that can function as cytokine receptors, including full-length or partial fragments with cytokine receptor function.

[0172] "Immune cell regulatory factors" refers to intracellular protein fragments in STAR that can function as immune cell regulatory factors, including full-length or partial fragments with immune cell regulatory factor functions.

[0173] The term "pharmaceutically acceptable" as used in this invention refers to the biological activity and characteristics of the active substances in the applied product that neither significantly stimulate the organism nor inhibit it.

[0174] The “…method” described in this invention can be used for the diagnosis and treatment of diseases, or for the diagnosis and treatment of non-disease purposes.

[0175] "The constant region of TCR" refers to the constant region and transmembrane fragments of the T cell receptor from animals such as humans or mice, including the constant region selected from the TCRα chain, the constant region of the TCRβ chain, the constant region of the TCRγ chain, and the constant region of the TCRδ chain. The "variants of the constant region of TCR" are variants that retain the functions of each constant region.

[0176] The term "comprising" or "including" as used in this invention is an open-ended expression. When used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the same or similar activity as the original sequence.

[0177] The term "homology" as used in this invention refers to the ability of those skilled in the art to adjust the sequence according to actual work needs when using protein or nucleotide sequences, so that the sequence used has (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39% homology compared to sequences obtained by existing technologies. %, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% of the sameness.

[0178] The term "treatment" as used in this invention refers to slowing down, interrupting, preventing, controlling, stopping, alleviating, reducing, or reversing a sign, symptom, disorder, condition, or progression or severity of a disease after it has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders.

[0179] The term "effective amount" as used in this invention refers to the amount or dose of the pharmaceutical composition of this invention that provides the desired treatment or prevention after being administered to an individual or organ in one or more doses.

[0180] The term "prevention" as used in this invention refers to a method implemented to prevent or delay the occurrence of a disease, condition, or symptom in the body.

[0181] The term "diagnosis" as used in this invention refers to determining whether a patient has had a disease or condition in the past, at the time of diagnosis, or in the future, or determining the progression of a disease or its possible future progression.

[0182] The "individual" referred to in this invention can be a human or a non-human mammal, or a cell, tissue, or organ of a human or non-human mammal. The non-human mammal can be a wild animal, a zoo animal, an economically important animal, a pet, a laboratory animal, etc. Preferably, the non-human mammal includes, but is not limited to, pigs, cattle, sheep, horses, donkeys, foxes, raccoon dogs, minks, camels, dogs, cats, rabbits, mice (e.g., rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels), or monkeys, etc.

[0183] The term "and / or" as used in this invention encompasses all combinations of items connected by the term, and should be considered as if each combination had been individually listed herein. For example, "A and / or B" includes "A", "A and B", and "B". As another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C".

[0184] The beneficial effects of this invention include:

[0185] This invention provides a CLL1-binding nanobody, and also provides a CLL1-binding STAR, a STAR specifically binding to CLL1 and LILRB4, a STAR complex, and immune cells expressing STAR or the STAR complex, which can be used for disease treatment, specifically including:

[0186] (1) In this invention, 25 nanobodies were constructed and 19 nanobodies that could specifically bind to CLL1 were screened out. The nanobodies NCLLO2 and NCLLO5 were screened out by the affinity test of nanobodies, the competitive binding test of epitope BLI and the specificity test of MPA.

[0187] (2) 25 nanobodies were prepared into STAR. STAR has the advantages of both CAR-T and TCR-T. It has high affinity comparable to CAR-T cells and high signal transduction capacity and high sensitivity to antigens as TCR-T. At the same time, it avoids the defects of both CAR-T and TCR-T. It avoids the dependence of TCR-T on HLA and the severe self-activation in CAR-T, so it is not easy to be depleted.

[0188] (3) This invention evaluated the in vitro function of 25 STARs and screened out 19 STARs that have specific recognition and killing effects on CLL1 target cells. Combined with in vitro verification of different AML cell lines, it was found that NCLL02-STAR, NCLL05-STAR and NCLL06-STAR have better killing effects on different AML cell lines.

[0189] (4) This invention assembles NCLL02 or NCLL05 and the LILRB4-binding nanobody NLB14 with the constant region of the STAR molecule to construct a STAR that can simultaneously bind to CLL1 and LILRB4, showing greater potential in the treatment of relapsed and refractory AML. In vivo and in vitro functional evaluations showed that both NCLL02-NLB14-STAR and NCLL05-NLB14-STAR have cytotoxic effects on target cells. In in vivo experiments, the weight of mice in the treatment group did not decrease significantly, indicating that the STAR constructed in this invention, which binds to two targets, has significant efficacy and safety. Based on the high expression of CLL1 and LILRB4 in AML (MV4-11) and their high expression in myeloid immunosuppressive cells, the development of STAR-T targeting CLL1 and LILRB4 can provide a new immunocellular therapy strategy for AML (MV4-11) patients by killing tumor cells and improving the tumor microenvironment.

[0190] (5) The present invention further optimizes the structure of STAR that binds to the dual targets, specifically by optimizing the connection between CLL1 antibody and LILRB4 antibody and optimizing the connection of functional domains. The results show that different optimization methods all show the killing effect on target cells, and the most optimal structure of STAR is selected. Attached Figure Description

[0191] Figure 1(A): Affinity test results of CLL1 nanobody (NCLL02)-BLI.

[0192] Figure 1(B): Affinity test results of CLL1 nanobody (NCLL05)-BLI.

[0193] Figure 1(C): Affinity test results of CLL1 nanobody (NCLL06)-BLI.

[0194] Figure 1(D): Affinity test results of CLL1 nanobody (NCLL10)-BLI.

[0195] Figure 1(E): Affinity test results of CLL1 nanobody (NCLL18)-BLI.

[0196] Figure 2(A): MPA specificity detection of CLL1 nanobody (NCLL02).

[0197] Figure 2(B): MPA specificity detection of CLL1 nanobody (NCLL05).

[0198] Figure 3: Schematic diagram of the vector structure of STAR targeting CLL1, where LS represents the GM-CSF signal peptide, VHH(NCLLs) represents the nanobody that binds to CLL1, namely any one of NCLL01-NCLL25, Cβ represents the constant region of the TCRβ chain or its variant, Furin-P2A represents the linker peptide, Ca represents the constant region of the TCRα chain or its variant, and (G4S)3 is the linker.

[0199] Figure 4: Killing effect of CLL1-STAR on THP1 and THP-CLL1KO target cells.

[0200] Figure 5: Killing effect of CLL1-STAR on MV4-11 and MV4-11-CLL1KO target cells.

[0201] Figure 6(A): Specific lysis of MV4-11 target cells by different STARs at E:T = 0.6:1.

[0202] Figure 6(B): Specific lysis of OCI-AML3 target cells by different STARs at E:T = 0.6:1.

[0203] Figure 7: Schematic diagram of STAR structure targeting CLL1 and LILRB4, where LS represents GM-CSF signal peptide, Cβ represents the constant region of TCRβ chain or its variant, Furin-P2A represents linker peptide, Ca represents the constant region of TCRα chain or its variant, and (G4S)3 is linker.

[0204] Figure 8(A): In vitro killing experiment of NCLL02-NLB14-STAR, NLB14-STAR, and NCLL02-STAR on target cells MV4-11, LILRB4KO, and CLL1KO.

[0205] Figure 8(B): In vitro killing experiments of NCLL05-NLB14-STAR, NLB14-STAR, and NCLL05-STAR on target cells MV4-11, LILRB4KO, and CLL1KO.

[0206] Figure 9(A): Concentrations of IL-2, IFN-γ, and TNF-α in NCLLs-NLB14-STAR cells under the MV4-11 target cell system.

[0207] Figure 9(B): Concentrations of IL-2, IFN-γ, and TNF-α in NCLLs-NLB14-STAR under the MV4-11-LILRB4KO system.

[0208] Figure 9(C): Concentrations of IL-2, IFN-γ, and TNF-α secreted by NCLLs-NLB14-STAR in the MV4-11-CLL1KO system.

[0209] Figure 10: In vivo functional evaluation of NCLLs-NLB14-STAR-T cells in mice with a CLL1 single-positive model (MV4-11-LUC-LILRB4KO target cell tumor model).

[0210] Figure 11: Weight evaluation of mice with NCLLs-NLB14-STAR-T cells in a CLL1 single-positive model (MV4-11-LUC-LILRB4KO target cell tumor model).

[0211] Figure 12: Evaluation of tumor fluorescence values ​​of NCLLs-NLB14-STAR-T cells in mice with a CLL1 single-positive model (MV4-11-LUC-LILRB4KO target cell tumor model).

[0212] Figure 13: In vivo functional evaluation of NCLLs-NLB14-STAR-T cells in mice using a CLL1 / LILRB4 mixed model (three target cells: MV4-11-LUC, MV4-11-LUC-LILRB4KO, and MV4-11-LUC-CLL1KO).

[0213] Figure 14: Evaluation of mouse body weight in a CLL1 / LILRB4 mixed model (three target cells: MV4-11-LUC, MV4-11-LUC-LILRB4KO, and MV4-11-LUC-CLL1KO) using NCLLs-NLB14-STAR-T cells.

[0214] Figure 15: Evaluation of tumor fluorescence values ​​of NCLLs-NLB14-STAR-T cells in mouse tumors in a CLL1 / LILRB4 mixed model (three target cells: MV4-11-LUC, MV4-11-LUC-LILRB4KO, and MV4-11-LUC-CLL1KO).

[0215] Figure 16(A): Schematic diagram of the structure of (NCLL05-(G4S)3-NLB14)ba STAR-a(G4S)3OX40 in CLL1*LILRB4-STAR, where LS represents the GM-CSF signal peptide, (G4S)3 is the linker, Cβ represents the constant region of the TCRβ chain or a variant thereof, Furin-P2A represents the linker peptide, and Ca represents the constant region of the TCRα chain or a variant thereof.

[0216] Figure 16(B): Schematic diagram of the structure of (NLB14-(G4S)3-NCLL05)ba STAR-a(G4S)3OX40 in CLL1*LILRB4-STAR, where LS represents the GM-CSF signal peptide, (G4S)3 is the linker, Cβ represents the constant region of the TCRβ chain or a variant thereof, Furin-P2A represents the linker peptide, and Ca represents the constant region of the TCRα chain or a variant thereof.

[0217] Figure 16(C): Schematic diagram of the structure of (NCLL05-M218-NLB14)ba STAR-a(G4S)3OX40 in CLL1*LILRB4-STAR, where LS represents the GM-CSF signal peptide, M218 and (G4S)3 are linkers, Cβ represents the constant region of the TCRβ chain or a variant thereof, Furin-P2A represents the linker peptide, and Ca represents the constant region of the TCRα chain or a variant thereof.

[0218] Figure 16(D): Schematic diagram of the structure of (NLB14-M218-NCLL05)ba STAR-a(G4S)3OX40 in CLL1*LILRB4-STAR, where LS represents the GM-CSF signal peptide, M218 and (G4S)3 are linkers, Cβ represents the constant region of the TCRβ chain or a variant thereof, Furin-P2A represents the linker peptide, and Ca represents the constant region of the TCRα chain or a variant thereof.

[0219] Figure 16(E): Schematic diagram of the structure of (NCLL05-(EAAAK)3-NLB14)ba STAR-a(G4S)3OX40 in CLL1*LILRB4-STAR, where LS represents the GM-CSF signal peptide, (EAAAK)3 and (G4S)3 are linkers, Cβ represents the constant region of the TCRβ chain or a variant thereof, Furin-P2A represents the linker peptide, and Ca represents the constant region of the TCRα chain or a variant thereof.

[0220] Figure 16(F): Schematic diagram of the structure of (NLB14-(EAAAK)3-NCLL05)ba STAR-a(G4S)3OX40 in CLL1*LILRB4-STAR, where LS represents the GM-CSF signal peptide, (EAAAK)3 and (G4S)3 are linkers, Cβ represents the constant region of the TCRβ chain or a variant thereof, Furin-P2A represents the linker peptide, and Ca represents the constant region of the TCRα chain or a variant thereof.

[0221] Figure 17: STAR membrane expression results of optimized connection between NCLL and NLB14 in CLL1*LILRB4-STAR, specifically including (NCLL05-(G4S)3-NLB14)ba STAR-a(G4S)3OX40 (corresponding to Figure 16A), (NLB14-(G4S)3-NCLL05)ba STAR-a(G4S)3OX40 (corresponding to Figure 16B), (NCLL05-M218-NLB14)ba STAR-a(G4S)3OX40 (corresponding to Figure 16C), (NLB14-M218-NCLL05)ba STAR-a(G4S)3OX40 (corresponding to Figure 16D), (NCLL05-(EAAAK)3-NLB14)ba STAR-a(G4S)3OX40 (corresponding to Figure 16E), (NLB14-(EAAAK)3-NCLL05)ba STAR-a(G4S)3OX40 (corresponding to Figure 16F), NCLL05b-NLB14a (corresponding to the carrier in Figure 7) connection structure.

[0222] Figure 18(A): STAR-specific lysis of MV4-11 target cells with different linking modes between NCLL antibody and NLB14 antibody in CLL1*LILRB4-STAR at E:T = 0.3:1.

[0223] Figure 18(B): STAR specific lysis of MV4-11-LILRB4KO target cells with different linkage modes between NCLL antibody and NLB14 antibody in CLL1*LILRB4-STAR at E:T=0.6:1.

[0224] Figure 19(A): Schematic diagram of the structure of NCLL05-NLB14 STAR-a(G4S)3OX40 in CLL1*LILRB4-STAR-T cells, where LS represents the GM-CSF signal peptide, Cβ represents the constant region of the TCRβ chain or a variant thereof, Furin-P2A represents the linker peptide, Ca represents the constant region of the TCRα chain or a variant thereof, and (G4S)3 is the linker.

[0225] Figure 19(B): Schematic diagram of the structure of NCLL05-NLB14 STAR-abOX40 in CLL1*LILRB4-STAR-T cells, where LS represents the GM-CSF signal peptide, Cβ represents the constant region of the TCRβ chain or a variant thereof, Furin-P2A represents the linker peptide, and Ca represents the constant region of the TCRα chain or a variant thereof.

[0226] Figure 19(C): Schematic diagram of the structure of NCLL05-NLB14 STAR-ab(G4S)3OX40 in CLL1*LILRB4-STAR-T cells, where LS represents the GM-CSF signal peptide, Cβ represents the constant region of the TCRβ chain or a variant thereof, Furin-P2A represents the linker peptide, Ca represents the constant region of the TCRα chain or a variant thereof, and (G4S)3 is the linker.

[0227] Figure 20: Short-term killing efficiency of CLL1*LILRB4-STAR-T cells linked to OX40 using different connection methods against different target cells.

[0228] Figure 21: Continuous killing efficiency of target cells by CLL1*LILRB4-STAR-T cells linked to OX40 using different connection methods. Detailed Implementation

[0229] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0230] Example 1: Screening of nanobodies targeting CLL1

[0231] 1.1 Immunization of alpacas with human CLL1 protein

[0232] Healthy alpacas were immunized with 100 μg of commercially available human CLL1 protein extracellular domain, using adjuvants including complete Freund's adjuvant (CFA, Sigma) and incomplete Freund's adjuvant (IFA, Sigma). The expressed and purified human CLL1 protein extracellular domain was diluted with PBS and then mixed 1:1 with the corresponding adjuvant. The antigen and adjuvant were thoroughly mixed to form a stable emulsion. The antigen mixture was drawn into a syringe and injected subcutaneously at multiple points on the alpaca's neck, with 100-200 μL injected at each point.

[0233] 1.2 PBMC Separation

[0234] 1) Separate PBMCs in a biosafety cabinet, combine the blood from the anticoagulant tube into a 50mL centrifuge tube (30mL), add PBS to a total volume of 50mL, and mix gently.

[0235] 2) Take a new 50mL centrifuge tube and add Ficoll separation buffer, 15mL / tube. Spread the blood sample on the surface of the Ficoll buffer, 25mL / tube. The process should be steady to allow the Ficoll and blood to separate into layers and prevent mixing.

[0236] 3) Set the centrifuge speed to 0, maintain room temperature, centrifuge at 800g for 30 minutes. After centrifugation, remove the sample from the centrifuge. The sample layers are as follows: upper aqueous phase - white film layer - Ficoll layer - red blood cell layer, where PBMCs are in the white film layer. Aspirate the white film layer and transfer it to a new 50mL centrifuge tube.

[0237] 4) Add PBS to 50 mL in the sample tube, mix well, centrifuge at 2000 rpm at room temperature for 5 min, discard the supernatant, and resuspend the cell pellet in 5 mL of PBS.

[0238] 5) Repeat step 4) and cool down to 4℃.

[0239] 6) Resuspend the cell pellet in 5 mL of PBS, add PBS to make up to 40 mL, and count the cells.

[0240] 7) Centrifuge at 1500 rpm, 4℃ for 5 min, discard the supernatant, add 1 mL PBS, resuspend the cells, and mix well; add 20 mL Trizol, mix well, incubate at room temperature for 5 min, lyse the cells, aliquot 1 mL into RNase-free 1.5 mL EP tubes, freeze at -80℃ for RNA extraction.

[0241] 1.3 RNA extraction and reverse transcription

[0242] 1) Remove the sample from -80℃, thaw at room temperature, add 200μL of chloroform, and let stand at room temperature for 3 min. Centrifuge the sample at 12000g for 15 min at 4℃. The sample will separate into an upper aqueous phase, an intermediate layer, and an organic layer. Transfer the upper layer to a new RNase-free tube, add 1μL of glycogen and 500μL of isopropanol, and let stand at 4℃ overnight.

[0243] 2) Centrifuge the sample at 12000g for 20 min at 4℃, remove the supernatant, add 1 mL of pre-cooled 75% ethanol to wash the precipitate, centrifuge again to remove the ethanol, and air dry. Add 15 μL of RNase-free water to each tube to dissolve the RNA precipitate and perform reverse transcription.

[0244] 3) cDNA was synthesized using the Promega reverse transcription kit (20 μL system).

[0245] Step 1: Take a certain amount of template RNA and add Oligo(dT), see Table 1;

[0246] Table 1

[0247] Step 2: Place the mixture of template RNA and Oligo(dT) at 65°C for 5 minutes for pre-denaturation, and then return it to ice.

[0248] Step 3: During pre-denaturation, RT-Mix can be prepared in advance, 8 μL per tube. The components and volumes are shown in Table 2.

[0249] Table 2

[0250] Step 4: Set up the reverse transcription program, including extension and reverse transcriptase inactivation. Once the program is complete, cDNA is obtained.

[0251] 1.4 Phage Library Construction

[0252] 1) Obtain the VHH sequence by PCR

[0253] The VHH sequence was obtained through two rounds of PCR, and homologous arms of the vector were added to both ends of the sequence.

[0254] 2) First round of PCR

[0255] Step 1: Prepare the reaction system as shown in Table 3.

[0256] Table 3

[0257] Step 2: PCR conditions are shown in Table 4.

[0258] Table 4

[0259] The PCR products were subjected to gel electrophoresis, and the target band at 0.7 kb was excised and the product was recovered.

[0260] 3) Second round of PCR

[0261] Step 1: Prepare the reaction system as shown in Table 5.

[0262] Table 5

[0263] Step 2: PCR conditions are shown in Table 6.

[0264] Table 6

[0265] The PCR products were subjected to gel electrophoresis, and the target band at 400 bp was excised and the product was recovered.

[0266] 4) Vector PCR

[0267] The vector region of phagemid was obtained by PCR and used to express the VHH sequence.

[0268] Step 1: Prepare the reaction system as shown in Table 7.

[0269] Table 7

[0270] Step 2: PCR conditions are shown in Table 8.

[0271] Table 8

[0272] The PCR products were subjected to gel electrophoresis, and the 4000bp target band was excised and the product was recovered.

[0273] 5) Ligation, purification and concentration of ligation products

[0274] The VHH fragment was ligated to the phagemid vector, and the ligation product was then concentrated.

[0275] Step 1: Prepare the reaction system as shown in Table 9.

[0276] Table 9

[0277] Step 2: Incubate the above mixture at 50°C for 2 hours, then cool it on ice.

[0278] Step 3: Purify the ligation product, remove salt ions and protein components from the ligation system, and concentrate the volume to 1 / 10 of the original volume.

[0279] 1.5. Power-to-database construction

[0280] 1) Take a tube of competent E. coli cells and thaw them on ice.

[0281] 2) Take 2 μL of the ligation product or positive control and add it to the competent cells above, then gently mix. Let it stand on ice for 1-2 min, then transfer it to a pre-cooled electroporation cuvette for electroporation.

[0282] 3) Immediately after electroporation, add 1 mL of 37℃ 2YT-G medium, rinse the electroporation cup with a pipette tip, and transfer the electroporated bacterial culture to a 15 mL centrifuge tube or a 2 mL EP tube. Incubate at 37℃ until all samples have been electroporated. (2YT-G: 2×YT medium containing 2% glucose) Transfer to a 37℃ shaker and incubate at 220 rpm for 1 h.

[0283] 4) Take 5 μL of the above bacterial solution and dilute it 10... 2 -10 5 Dilute the sample with 2YT-A (2YT plate containing 100 μg / mL ampicillin) and incubate overnight at 37°C for colony counting.

[0284] 5) Inoculate the remaining bacterial culture into 2YT-AG medium, shake to the logarithmic growth phase, add helper phages for infection, and incubate at 30°C and 220 rpm for 12-16 hours. (2YT-AG: 2×YT medium containing 2% glucose and 100 μg / mL ampicillin)

[0285] 6) Collect and concentrate the phages, and determine the titer.

[0286] 1.6. Phage library antibody screening

[0287] The phage library obtained above was subjected to three rounds of antibody screening, each round including a positive selection and a negative selection. First, the phages were incubated with the antigen peptide; phages that could not bind were discarded, leaving only the phages that bound the antigen peptide. Then, the phages were incubated with BSA for negative selection, leaving only the phages that could not bind to BSA.

[0288] 1) Coat plate. Dilute the antigen to a concentration of 2 ng / μL with PBS and add it to a 96-well plate at 100 μL / well; prepare 2% BSA with PBS and add it to the corresponding negative wells at 100 μL / well. Seal with plastic wrap and incubate overnight at 4°C.

[0289] 2) Discard the coating solution, add 200 μL of washing buffer (washing buffer: 1% Tween 20 / PBS, pH 7.4), and wash 3 times.

[0290] 3) Sealing. Add 2% BSA blocking solution to all wells, 100 μL / well, seal with plastic wrap, and incubate at 37°C for 1 h.

[0291] 4) Discard the supernatant, add 200 μL of washing solution, and wash 3 times.

[0292] 5) Add the bacteriophage to the cation wells, 1×10 12 phages / well, diluted to 100 μL, sealed with plastic wrap, and incubated at 37°C for 1 hour.

[0293] 6) Discard the supernatant, add 200 μL of washing solution, and wash 10 times.

[0294] 7) Elution-Neutralization. Add 200 μL of eluent to the sun-facing well and neutralize to pH 7-7.4.

[0295] 8) Anion selection. Add the above eluent to the anion selection well, seal with plastic wrap, incubate at 37°C for 1 hour, collect and retain the supernatant, and perform titer detection.

[0296] 9) Take a small amount of phage after one round of panning, dilute it, and spread it on a 2×YT-A plate. Incubate at 37°C overnight. The next day, count the colonies and calculate the titer. Select single clones for sequencing to analyze sequence diversity and enrichment.

[0297] 10) The remaining bacteriophages were all used for TG1 infection.

[0298] 11) M13KO7 infection. Dilute the phage, add M13KO7 to the bacterial culture, and incubate at 37°C for 30 min; replace with 2×YT-AK medium, and incubate at 30°C and 220 rpm for 14-16 h.

[0299] 12) Concentrate the phage and detect the phage titer, then proceed to the next round of screening.

[0300] The amount of coating antigen is reduced in the second and third rounds of screening, and the number of washing cycles is increased after the phage is incubated with the positive wells. Other steps are the same as those described above.

[0301] 1.7. Obtaining results by combining detection and sequencing.

[0302] The phages obtained from the three rounds of screening were co-infected with TG1 with M13KO7 helper phages, plated on 2YT-AK plates, and single clones were picked for phage amplification. The phages were collected for binding detection to determine the usable phages / antibodies.

[0303] 1) Coat plate. Dilute the antigen to 1 ng / μL with coating buffer, 100 μL / well; add 2% BSA to the control negative well; seal with plastic wrap and incubate overnight at 4°C.

[0304] 2) Discard the coating solution in the plate and wash 3 times with 200-250 μL of washing solution.

[0305] 3) Add 200 μL of 2% BSA to all wells and seal at room temperature for 1 h.

[0306] 4) Discard the blocking solution in the plate, add 200 μL of washing solution, and wash once.

[0307] 5) Add 100 μL of phage to each of the positive and negative wells and incubate at 37°C for 1 h.

[0308] 6) Discard the phages in the plate, add 200 μL of washing buffer, and wash 3 times.

[0309] 7) Dilute the anti-M13-HRP antibody at 50 ng / well and incubate at room temperature for 1 hour.

[0310] 8) Discard the antibody from the plate, add 200 μL of washing buffer, and wash 5 times.

[0311] 9) Add 100 μL of TMB colorimetric solution to each well and react at room temperature until the OD value is between 2 and 3.

[0312] 10) Add stop solution to the color development system, 50 μL per well.

[0313] 11) Measure the absorbance at 450 nm using a spectrophotometer. Send the monoclonal bacterial culture corresponding to the positive well for sequencing to determine the VHH sequence.

[0314] 12) The obtained antibodies were named NCLL01-NCLL25 respectively.

[0315] Example 2: CLL1 nanobody BLI affinity detection

[0316] Antibody affinity is determined using biomembrane interference (BLI) technology, utilizing Fortebio The detection instrument determined the antibody affinity. The KD value for each sample was calculated using the Octet RED96e (ForteBio) Data Analysis 11 software. The affinity detection results for the CLL1 nanobody BLI are shown in Table 10 and Figures 1(A)-1(E). Table 10 shows the single-concentration detection results of CLEC12A Protein, Human, Recombinant (ECD, His Tag) and antibody, where the ligand concentration was 10 μg / mL, the ligand immobilization level on the sensor chip was 3.0 nm, the Fit Model was 1:1 binding, and the results showed R... 2 All are greater than 0.99.

[0317] Table 10: CLL1 nanobody BLI affinity assay

[0318] The Fc fragment sequence is shown in SEQ ID NO: 163, and the NCLLs and Fc are connected by G4S.

[0319] Example 3: CLL1 nanobody epitope BLI competitive binding assay

[0320] Competitive binding of antibodies is achieved via biomembrane interference (BLI) technology, utilizing Fortebio The detection was performed using advanced instrument technology. The stationary phase was CLL1 protein, and the mobile phase consisted of NCLL02-Fc, NCLL05-Fc, NCLL06-Fc, NCLL10-Fc, or NCLL18-Fc. The results of the antibody epitope competitive inhibition rate assay for the CLL1 nanobody using BLI are shown in Table 11. Table 12 shows the competitive inhibition rate minus the self-reaction signal from Table 11. The criteria were: 70%-100% for no competition, 30%-70% for partial competition, and <30% for complete competition. The results showed that NCLL02-Fc, NCLL05-Fc, and NCLL06-Fc exhibited complete competitive binding to CLL1. The sequences are shown in Table 13.

[0321] Table 11: Results of antibody epitope competition inhibition rate in CLL1 nanobody BLI affinity assay

[0322] Table 12: Results of Antibody Epitope Competition Inhibition Rate

[0323] Table 13: CDR amino acid sequence of CLL1 nanobody

[0324] The amino acid sequence of antibody NCLL02 is shown in SEQ ID NO: 25, the amino acid sequence of antibody NCLL05 is shown in SEQ ID NO: 26, and the amino acid sequence of antibody NCLL06 is shown in SEQ ID NO: 27.

[0325] Example 4: MPA Specificity Detection of CLL1 Nanobody

[0326] The Membrane Proteome Array (MPA) developed and used by Integral Molecular is an array of over 6,000 human membrane proteins (covering 94% of human membrane proteins). Each human membrane protein in the MPA has a complete structure and can be expressed in its native conformation within living cells. The MPA is an in vitro tool that enables rapid and comprehensive screening of the specificity of candidate therapeutic drug compositions. As shown in Figures 2(A)-2(B), no off-target effects were detected by the MPA assay for the NCLL02-Fc and NCLL05-Fc antibodies in this embodiment.

[0327] Example 5: Anti-CLL1-STAR structure

[0328] In this invention, STAR function is improved by using reported methods such as murine derivatization of the constant region, point mutation of cysteine, and hydrophobic amino acid mutation of the α chain constant region.

[0329] Humanization / mouse-derived modification of constant regions: Since the constant region sequences of human, primate and mouse TCRα / β chains (mouse TCRAC / mouse TCRBC) are highly conserved in function and have the same key amino acid sequences, they can be substituted for each other. After substitution, the efficiency of correct pairing of STAR molecules is increased on the one hand, and the possibility of mismatches causing unknown specificity is reduced on the other hand, thus increasing safety.

[0330] 1) Modifications derived from mouse homeostatic regions

[0331] The constant region sequence of the mouse-derived TCRα / β chain can be modified by mouse-derived modification, cysteine ​​substitution, and transmembrane domain hydrophobic amino acid substitution, as shown in Table 14.

[0332] Secondly, point mutations of cysteine ​​residues are introduced into disulfide bonds: a threonine T mutation at position 48 is replaced with a cysteine ​​C mutation in the constant region of the murine TCR α chain, and a serine S mutation at position 56 is replaced with a cysteine ​​C mutation in the constant region of the murine TCR β chain. These two newly added cysteine ​​residues will form disulfide bonds between the two STAR chains, reducing mismatches between the two STAR chains and the endogenous TCR chain, and helping the STAR molecule form a more stable complex.

[0333] The STAR transmembrane region was designed with hydrophobic amino acid substitutions: Mutations were made at three amino acid sites within the transmembrane region of the TCR α chain's constant region, from amino acid positions 111 to 119. Serine (S) at position 112 was replaced with leucine (L), methionine (M) at position 114 with isoleucine (I), and glycine (G) at position 115 with valine (V). The overall amino acid sequence in this region changed from LSVGMLRIL to LLVIVLRIL. This design increased the hydrophobicity of the transmembrane region, counteracting the instability caused by the positive charge carried by the TCR transmembrane region, allowing the STAR molecule to exist more stably on the cell membrane and thus acquire better function.

[0334] To further optimize the design of the STAR molecule, a specific rearrangement was performed on the N-terminus of the STAR molecule's constant region, based on murine derivatization of the constant region, cysteine ​​point mutations, and hydrophobic amino acid mutations in the α-chain constant region, to achieve better results. Rearrangement involves partial sequence deletion and humanization mutation of other sequences. The significance of humanization mutation lies in minimizing non-human sequences in the STAR molecule while maintaining its function, thereby minimizing the possibility of receptor rejection of STAR-T cells in clinical applications. Therefore, the N-terminus of the TCR α-chain constant region was further modified, including replacing amino acid E at position 6 with D, replacing K at position 13 with R, and deleting amino acids 15-18. The resulting α-chain constant region was named TRAC (Nrec-Cys-TM). Further modifications were made to the N-terminal 25 amino acids of the TCR β-chain constant region, including replacing the 3rd amino acid (R) with K, the 6th amino acid (T) with F, the 9th amino acid (K) with E, the 11th amino acid (S) with A, and the 12th amino acid (L) with V, and the 17th and 21-25th amino acids were deleted. The resulting β-chain constant region was named TRBC (Nrec-Cys-TM).

[0335] Table 14: Amino acid mutations / modifications in the TCR constant region of mice from this source

[0336] 2) Modifications derived from human constant regions

[0337] For the constant region sequence of the human TCRα / β chain, one or more of the following modifications can be performed: mouse derivatization, cysteine ​​substitution, transmembrane domain hydrophobic amino acid substitution, or rearrangement, among which:

[0338] Table 15 shows the constant region sequences of human TCRα / β chains modified by a combination of mouse-derived modifications, cysteine ​​substitutions, and transmembrane domain hydrophobic amino acid substitutions.

[0339] Table 15: TCR constant region mutations / modifications from human sources

[0340] 3) Modification of co-stimulatory domains

[0341] Furthermore, the inventors modified the αβ-STAR with co-stimulatory signals to enhance the performance of αβT cells. The function of STAR can be further enhanced by linking cytoplasmic regions of functional domains preferably co-stimulatory molecules such as OX40 (amino acid sequence as SEQ ID NO: 125: RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI), CD40 (amino acid sequence as SEQ ID NO: 126), ICOS (amino acid sequence as SEQ ID NO: 127), CD28 (amino acid sequence as SEQ ID NO: 128), 4-1BB (amino acid sequence as SEQ ID NO: 129), or CD27 (amino acid sequence as SEQ ID NO: 130) to the C-terminus of the α-chain constant region, β-chain constant region, and α-chain constant region. The co-stimulatory molecules can be directly linked or linked to the C-terminus of the α-chain constant region and / or β-chain constant region via a linker (e.g., to a (G4S)3 linker). In addition to the modifications described above, the constant region linked to the co-stimulatory molecule may also lack the native intracellular region relative to the wild-type constant region, which further improves STAR function. For example, the α-chain constant region may lack amino acids 136-137; and / or, the β-chain constant region may lack amino acids 167-172.

[0342] In some specific embodiments, the sequence of the constant region of the TCRα chain includes any one of SEQ ID NO: 28, 30, 106-113.

[0343] In some specific embodiments, the sequence of the constant region of the TCRβ chain includes any one of SEQ ID NO: 29, 31, 114-119;

[0344] In some specific embodiments, the amino acid sequence of the constant region of the TCRγ chain includes SEQ ID NO: 32 or 104.

[0345] In some specific embodiments, the amino acid sequence of the constant region of the TCRδ chain includes SEQ ID NO: 33 or 105.

[0346] The antigen-binding region in the STAR structure includes the nanobodies NCLL01-NCLL25 screened in Example 1. For example, at least one nanobodies NCLL01-NCLL25 may be attached to the N-terminus of the TCRβ chain constant region or a variant thereof, or at least one nanobodies NCLL01-NCLL25 may be attached to the N-terminus of the TCRα chain constant region or a variant thereof. Alternatively, at least one nanobodies NCLLs may be attached to both the TCRα chain constant region or a variant thereof and the N-terminus of the TCRβ chain constant region or a variant thereof.

[0347] Example 6: Construction of Anti-CLL1-STAR vector and viral packaging

[0348] 1) Construct a STAR structure targeting CLL1 (Figure 3), NCLLs(b)-STAR-a(G4S)3-OX40 (abbreviated as NCLLs-STAR). Assemble the positive antibody VHH sequence (NCLL01-NCLL25) obtained from the previous screening with the constant region of the STAR molecule (see Example 5), and use homologous recombination to insert it into a lentiviral vector to construct a complete STAR plasmid.

[0349] 2) Packaging viruses

[0350] Lentix-293T cells were divided into groups of 5 × 10 5 Cells / mL were inoculated into 10cm culture dishes and cultured in a 37℃, 5% CO2 incubator. Transfection was performed when the cell density reached approximately 80% (observed under a microscope). The four plasmids were mixed with 500μL of serum-free DMEM at a ratio of PMD2.G:PRSV-Rev:PMDlg:transfer plasmid = 1:1:2:4. 54μL of PEI-max was mixed with 500μL of serum-free DMEM and incubated at room temperature for 5 min (PEI-Max to plasmid volume ratio 3:1). The PEI-max mixture was slowly added to the plasmid mixture, gently pipetting to mix, and incubated at room temperature for 15 min. The final mixture was slowly added to the culture medium, thoroughly mixed, and then returned to the incubator for 12-16 h of further culture. The culture was then transferred to 6% FBSDMEM medium, and virus culture was collected at 48 h and 72 h.

[0351] 3) Virus titer measurement

[0352] Jurkat-C4 cells with TCR knockout were used at 1.5 × 10⁻⁶ 5 Cells were seeded at a rate of 100 μL / mL in 96-well plates. Each well contained 100 μL of 1640 medium with 10% FBS and 0.2 μL of 1000× polybrene. For virus dilution, 10-fold serial dilutions were performed using complete 1640 medium. The diluted cells were added to the virus wells at a rate of 100 μL / well, mixed, centrifuged at 32°C and 1500 rpm for 90 min, and then incubated at 37°C with 5% CO2. After 72 h, the infection efficiency was measured by flow cytometry. When calculating the titer, wells with an infection rate of 2-30% were selected. The calculation formula is: Titer (TU / mL) = 1.5 × 10⁻⁶. 4 ×Positive rate ÷Viral volume (μL) × 1000. Infect T cells with the above virus to express STAR.

[0353] 4) Isolation, activation and infection of human primary T cells

[0354] After thawing, frozen PBMCs were cultured in X-VIVO medium containing 10% FBS and 10 ng / mL IL-7, 10 ng / mL IL-15, and 5 ng / mL IL-21, with an initial culture density of 1×10⁻⁶. 6 The virus solution was added to the culture medium at a concentration of / mL, along with the novel T-cell activation reagent T Cell TransAct conjugated with humanized CD3 and CD28 antibodies for activation. After 24 hours of activation, the virus solution was added, mixed well, and incubated in a CO2 incubator. After 48 hours of infection, X-VIVO medium containing 10% FBS and 10 ng / mL IL-7, 10 ng / mL IL-15, and 5 ng / mL IL-21 was added and the culture was transfected into wells. Subculture was performed every 1-2 days thereafter.

[0355] Example 7: In vitro functional evaluation of Anti-CLL1-STAR

[0356] Luciferase is a common substance used in cell function research. Enzyme activity is determined by adding a luciferase substrate to a system, and luciferase activity is closely related to the expression and binding strength of the target gene, as well as the number of cells. In this invention, a target cell line stably expressing luciferase is established, and the amount of luciferase is used to indicate the number of target cells, thereby indicating the cytotoxic function of functional cells.

[0357] 7.1 In vitro screening of CLL1 nanobodies

[0358] In Experiment 1, the NCLLs-STAR vector (see Example 6) was expressed in T cells. The positive control group (PC) used CAR-T cells containing the M26 antibody targeting CLL1 (amino acid sequence as shown in SEQ ID NO: 142). THP1 and THP1-CLL1KO target cells expressing CLL1 were seeded in 24-well plates at a density of 4E5 cells / well. STAR-T cells were added to the target cells at a 1:1 ratio of STAR-positive T cells to target cells, with a co-culture volume of 1 mL. After 24 hours of co-culture, the co-cultured cell suspension was collected, and the LUC (luciferase-reporter gene) luminescence value was detected using a luciferase reporter gene assay kit to calculate the killing efficiency of STAR-T cells against target cells. The experimental results are shown in Figure 4.

[0359] In Experiment 2, the NCLLs-STAR vector (see Example 6) was expressed in T cells. MV4-11 and MV4-11-CLL1KO target cells expressing the CLL1 target were seeded in 24-well plates at a density of 4E5 cells / well. STAR-T cells were added to the target cells at a 1:1 ratio of STAR-positive T cells to target cells, with a co-culture volume of 1 mL. After 24 hours of co-culture, the co-cultured cell suspension was collected, and the LUC (luciferase-reporter gene) luminescence value was detected using a luciferase reporter gene assay kit to calculate the killing efficiency of STAR-T cells against target cells. The experimental results are shown in Figure 5.

[0360] Experimental Results: As shown in Figure 4, for THP1 and THP1-CLL1KO target cells: NCLL02-STAR, NCLL05-STAR, NCLL06-STAR, and NCLL10-STAR exhibited specific killing and recognition of THP1 target cells; NCLL18-STAR did not recognize THP1 target cells. As shown in Figure 5, for MV4-11 and MV4-11-CLL1KO target cells: NCLL02-STAR, NCLL05-STAR, and NCLL06-STAR exhibited specific killing and recognition of MV4-11 target cells; NCLL10-STAR exhibited non-specific recognition and killing of MV4-11 target cells; NCLL18-STAR did not recognize MV4-11 target cells.

[0361] In summary, NCLL02-STAR, NCLL05-STAR, and NCLL06-STAR exhibit specific killing and recognition of CLL1 target cells for all the aforementioned target cells; the first and second peptide chain sequences of STARs that have recognition and killing effects on all target cells are shown in Table 16.

[0362] Table 16

[0363] 7.2 CLL1-STAR in vitro killing of different AML cell lines

[0364] In Experiment 3, CAR-T cells containing the NCLLs-STAR vector (see Example 6) expressed in T cells as described in Experiment 2, and the positive control group (PC) used CAR-T cells containing the M26 antibody targeting CLL1 (amino acid sequence as shown in SEQ ID NO: 142). Different AML cell lines, MV4-11 and OCI-AML3, were used as target cells. MV4-11 and OCI-AML3 target cells were seeded in 24-well plates at a density of 4E5 cells / well. STAR-T cells were added to the target cells at a ratio of 0.6:1 (STAR ​​positive T cells to target cells), with a co-culture volume of 1 mL. After 24, 48, and 72 hours of co-culture, the co-cultured cell suspension was collected, and the LUC luminescence value was detected using a luciferase reporter gene assay kit to calculate the killing efficiency of STAR-T cells against target cells. Figures 6(A)-(B) show that NCLL02-STAR, NCLL05-STAR, and NCLL06-STAR all exhibit good killing effects on MV4-11 and OCI-AML3 target cells.

[0365] Example 8: Construction of CLL1*LILRB4-STAR vector and viral packaging

[0366] The CLL1 nanobody NCLL02 or NCLL05 sequence and the LILRB4 nanobody were assembled with the STAR molecule constant region (see Example 5) and inserted into a lentiviral vector using homologous recombination to construct a complete CLL1*LILRB4-STAR plasmid.

[0367] The LILRB4 nanobody comprises NLB4 and NLB14, wherein:

[0368] The amino acid sequence of CDR1 in NLB4 is SEQ ID NO: 10: GTSGNVKAVG;

[0369] The amino acid sequence of CDR2 in NLB4 is SEQ ID NO: 11: TITRGGIPN;

[0370] The amino acid sequence of CDR3 in NLB4 is SEQ ID NO: 12: RILTDDWHDL;

[0371] The amino acid sequence of NLB4 is SEQ ID NO: 121;

[0372] The nucleotide sequence of NLB4 is SEQ ID NO: 123;

[0373] The amino acid sequence of CDR1 in NLB14 is SEQ ID NO: 13: GFTLDYYAIG;

[0374] The amino acid sequence of CDR2 in NLB14 is SEQ ID NO: 14: CVSSSDGSTY;

[0375] The amino acid sequence of CDR3 in NLB14 is SEQ ID NO: 15: DQYSSTWTIRLTRCHFGS;

[0376] The amino acid sequence of NLB14 is SEQ ID NO: 120;

[0377] The nucleotide sequence of NLB14 is SEQ ID NO: 122.

[0378] Taking NLB14 as an example, Figure 7 shows the structure for constructing a STAR structure targeting CLL1*LILRB4: NCLLs(b)-NLB14(a)-STAR-a(G4S)3-OX40 (abbreviated as NCLLs-NLB14-STAR). The first peptide chain sequence of NCLL02(b)-NLB14(a)STAR-a(G4S)3OX40 is shown in SEQ ID NO: 143, and the second peptide chain sequence is shown in SEQ ID NO: 144. The first peptide chain sequence of NCLL05(b)-NLB14(a)STAR-a(G4S)3OX40 is shown in SEQ ID NO: 145, and the second peptide chain sequence is shown in SEQ ID NO: 146.

[0379] In addition to the connection method shown in Figure 7, the positions of the CLL1-binding antibody and the LILRB4-binding antibody can be interchanged. Alternatively, the CLL1-binding antibody and the LILRB4-binding antibody can be tandemly linked to the constant region of the TCRα chain or a variant thereof, or to the constant region of the TCRβ chain or a variant thereof. Alternatively, both the constant region of the TCRα chain or a variant thereof (see Example 5) and the constant region of the TCRβ chain or a variant thereof (see Example 5) can be linked to the sequence of the tandemly linked CLL1-binding antibody and LILRB4-binding antibody. The tandem linking of the CLL1-binding antibody and the LILRB4-binding antibody can be, from the N-terminus to the C-terminus, the antibody binding CLL1 and the antibody binding LILRB4, or vice versa.

[0380] Example 9: In vitro functional evaluation of CLL1*LILRB4-STAR-T

[0381] Luciferase is a common substance used in cell function research. Enzyme activity is determined by adding a luciferase substrate to a system, and luciferase activity is closely related to the expression and binding strength of the target gene, as well as the number of cells. In this invention, a target cell line stably expressing luciferase is established, and the amount of luciferase is used to indicate the number of target cells, thereby indicating the cytotoxic function of functional cells.

[0382] In Experiment 1, the vectors NCLL02-STAR (see Example 6), NCLL05-STAR (see Example 6), NLB14-STAR (obtained by replacing NCLLs with NLB14 in the vector of Example 6), NCLL02-NLB14-STAR (see Figure 7), or NCLL05-NLB14-STAR (see Figure 7) were expressed in T cells, and uninfected STAR T cells (MOCK-T) were used as a control. MV4-11, MV4-11-LILRB4KO, and MV4-11-CLL1KO target cells were seeded in 24-well plates at a density of 4E5 / well. The corresponding number of STAR-T cells were added to the target cells at a ratio of 0.3:1 (STAR-positive T cells to target cells), with a co-culture volume of 1 mL. After 24 hours of co-culture, the co-cultured cell suspension was collected, and the LUC luminescence value was detected using a luciferase reporter gene assay kit to calculate the killing efficiency of STAR-T cells against target cells. As shown in Figures 8(A)-8(B), NCLL02-NLB14-STAR and NCLL05-NLB14-STAR have the ability to kill LILRB4 and CLL1 single-positive and double-positive target cells.

[0383] In Experiment 2, the NCLL02-NLB14-STAR (vector shown in Figure 7) or NCLL05-NLB14-STAR (vector shown in Figure 7) vectors were expressed in T cells, and uninfected STAR T cells (MOCK-T) were used as a control. Target cells expressing CLL1 and LILRB4 targets, namely MV4-11, MV4-11-LILRB4KO, and MV4-11-CLL1KO, were seeded in 24-well plates at a density of 4E5 / well. The corresponding number of STAR-T cells were added to the target cells at a ratio of 0.3:1 (STAR-positive T cells to target cells). The co-culture volume was 1 mL. After co-culturing for 24 hours, the co-cultured cell suspension was collected, and the supernatant was used to detect cytokines. As shown in Figures 9(A)-(C), when co-cultured with MV4-11-LILRB4KO target cells, NCLL05-NLB14-STAR cytokines were superior to NCLL02-NLB14-STAR, while when co-cultured with MV4-11 and MV4-11-CLL1KO target cells, the cytokines of NCLL02-NLB14-STAR or NCLL05-NLB14-STAR were comparable.

[0384] Example 10: In vivo functional evaluation of CLL1*LILRB4-STAR-T

[0385] 10.1 Experimental animal models

[0386] An NPG immunodeficient mouse model was established using NPG mice. These mice lack T cells, B cells, and NK cells, and also exhibit deficiencies in macrophages and dendritic cells. Female NPG mice aged 6–8 weeks were used in this experiment, with the weight difference between batches controlled to within 2g. Mice were housed in individually ventilated cages in specific pathogen-free (SPF) environments, provided with a normal diet and slightly acidic drinking water to prevent pathogen contamination.

[0387] 10.2 Animal Experiment Procedures

[0388] All animal procedures were performed only after the Animal Protocol was approved.

[0389] 10.3 Functional evaluation of CLL1*LILRB4-STAR-T cells in a CLL1 monopositive model in vivo

[0390] To verify the cytotoxic effect of STAR-T cells in vivo and to investigate potential safety issues, a MV4-11-LUC-LILRB4KO target cell tumor model was constructed. The effects of NCLL02-NLB14-STAR (vector shown in Figure 7), NCLL05-NLB14-STAR (vector shown in Figure 7), and uninfected STAR T cells (MOCK-T) on in vivo efficacy and expansion were studied. Fluorescently labeled MV4-11-LUC-LILRB4KO cells were reinfused into 6-8 week old female NPG mice via tail vein at a dose of 2E6 per mouse. On day 6 post-infusion, NCLL02-NLB14-STAR (vector shown in Figure 7), NCLL05-NLB14-STAR (vector shown in Figure 7), and uninfected STAR T cells (MOCK-T) were infused via tail vein at a dose of 2E6 per mouse. Then, on days -1, 6, 9, 14, 20, 33, and 41, tumor growth, tumor fluorescence values, and weight changes were detected using a luciferin substrate-catalyzed luminescence assay. The results are shown in Figures 10, 11, and 12. NCLL02-NLB14-STAR-T and NCLL05-NLB14-STAR-T showed good tumor inhibition against CLL1+ target cells, and neither type of T cell resulted in a significant decrease in mouse weight, indicating relatively good safety.

[0391] 10.4 Functional evaluation of CLL1*LILRB4-STAR-T cells in CLL1*LILRB4 mixed model mice

[0392] To verify the cytotoxic effect of STAR-T cells in vivo and to address potential safety issues, three target cell hybrid models were constructed: MV4-11-LUC, MV4-11-LUC-LILRB4KO, and MV4-11-LUC-CLL1KO, with proportions of 20%, 70%, and 10%, respectively. Six STAR-T cell groups were selected: NCLL02-NLB14-STAR (vector shown in Figure 7), NCLL05-NLB14-STAR (vector shown in Figure 7), NLB14-STAR (obtained by replacing NCLLs with NLB14 in the vector of Example 6), NCLL02-STAR (vector shown in Example 6), NCLL05-STAR (vector shown in Example 6), and uninfected STAR-T cells (MOCK-T) to study the effects on in vivo efficacy and in vivo expansion. Fluorescently labeled MV4-11-LUC (20%) + MV4-11-LUC-LILRB4KO (70%) + MV4-11-LUC-CLL1KO (10%) cells were injected intravenously into female NPG mice aged 6–8 weeks at a dose of 2E6 / mouse. On day 8 after tumor formation, the above six groups of STAR-T cells were infused intravenously at a dose of 3E6 / mouse. Tumor growth, tumor fluorescence values, and weight changes were then detected by luciferase substrate catalysis on days -1, 4, 7, 11, 14, and 19. The results are shown in Figures 13, 14, and 15. NCLL02-NLB14-STAR-T and NCLL05-NLB14-STAR-T showed good tumor inhibition effects on target cells. Moreover, the inhibitory effects of NCLL02-NLB14-STAR-T and NCLL05-NLB14-STAR-T on tumor cells were significantly better than those of NLB14-STAR, NCLL02-STAR, and NCLL05-STAR. Among them, NCLL05-NLB14-STAR-T and NCLL02-NLB14-STAR-T had comparable inhibitory effects on mixed CLL1 and LILRB4 tumor cells. Furthermore, the body weight of mice did not decrease significantly with either type of T cell, indicating relatively good safety.

[0393] Example 11: Functional validation of CLL1*LILRB4-STAR-T with different linking modes of NCLLs antibody and NLB14 antibody

[0394] To investigate the CLL1*LILRB4-STAR structure, the linker between the CCL1 antibody and the LILRB4 antibody was specifically analyzed. The CLL1*LILRB4-STAR structure shown in Figures 16(A)-16(F) and Table 17 was constructed. CLL1 and LILRB4 can be linked through (EAAAK)3linker (SEQ ID NO:36), (G4S)3linker (SEQ ID NO:68), and M218 linker (amino acid sequence SEQ ID NO:103, nucleotide sequence SEQ ID NO:141).

[0395] Table 17: Names of STARs used in the examples and amino acid sequence numbers of the two peptide chains

[0396] 11.1 Upper Membrane Expression

[0397] Primary T cells targeting CLL1*LILRB4-STAR with different linker linkages (Fig. 16: vectors (NCLLs-(linker)-NLB14)ba-STAR-a(G4S)3OX40 or (NBL14-(linker)-NCLLs)ba-STAR-a(G4S)3OX40) and Fig. 7: vector NCLL05(b)-NLB14(a)STAR-a(G4S)3OX40) were infected with lentiviruses, and their membrane expression was detected by flow cytometry. RFP fluorescent protein was co-expressed at the STAR terminal via IRES. The membrane expression of CLL1*LILRB4-STAR was determined by anti-mouse TCRβ chain antibody and RFP expression efficiency. The experimental results are shown in Fig. 17, indicating that CLL1*LILRB4-STAR with different linkage methods could be successfully membrane-bound.

[0398] 11.2. Killing efficiency of CLL1*LILRB4-STAR-T antibodies with different binding modes against different target cells using NCLLs and NLB14 antibodies.

[0399] MV4-11-LUC and MV4-11-LUC-LILRB4KO target cells expressing CLL1 and LILRB4 targets were seeded in 24-well plates at a density of 4E5 / well, as shown in Figure 16. Different structures were used, including CLL1*LILRB4-STAR-T (vector (NCLLs-(linker)-NLB14)ba-STAR-a(G4S)3OX40 or (NBL14-(linker)-NCLLs)ba-STAR-a(G4S)3OX40) and the vector NCLL05 (b) shown in Figure 7. STAR-T cells were used at STAR-positive T cell ratios of 0.3:1 with MV4-11-LUC target cells and 0.6:1 with MV4-11-LUC-LILRB4KO target cells. The corresponding number of STAR-T cells were added to the target cells, with a co-culture volume of 1 mL. After 24 hours of co-culture, the co-cultured cell suspension was collected, and the LUC luminescence value was detected using a luciferase reporter gene assay kit to calculate the killing efficiency of STAR-T cells against the target cells.

[0400] The experimental results are shown in Figures 18(A)-18(B). In MV4-11 and MV4-11-LILRB4KO target cells, STAR-T cells with different linking modes of NCLLs antibody and NLB14 antibody, namely CLL1*LILRB4-STAR-T (vector (NCLLs-(linker)-NLB14)ba-STAR-a(G4S)3OX40 or (NBL14-(linker)-NCLLs)ba-STAR-a(G4S)3OX40 or NCLL05(b)-NLB14(a)STAR-a(G4S)3OX40) all showed good killing effects on tumor cells.

[0401] Example 12: Functional verification of CLL1*LILRB4-STAR-T co-stimulatory domains connected in different ways

[0402] The co-stimulatory intracellular domain can be tandemly linked to the C-terminus of the TCR α chain, the C-terminus of the β chain, or the C-terminus of both the α and β chains (co-STAR). Furthermore, the OX40 domain of the co-stimulatory molecule can be directly linked to the C-terminus of the TCR constant region or via G4S (amino acid sequence SEQ ID NO: 66: GGGGS) or (G4S)3 (amino acid sequence SEQ ID NO: 68: GGGGSGGGGSGGGGS). See Figures 19(A)-19(C) for different structures of CLL1*LILRB4-STAR-T cells. The specific connection methods and sequence numbers are listed in Table 18.

[0403] Table 18

[0404] 12.1 Killing efficiency of CLL1*LILRB4-STAR-T cells with different co-stimulatory domain connections against different target cells

[0405] Target cells expressing CLL1 and LILRB4 targets, namely MV4-11-LUC, MV4-11-LUC-LILRB4KO, and MV4-11-LUC-CLL1KO, were seeded in 24-well plates at a density of 4E5 / well. As shown in Figures 19(A)-19(C), different structures of CLL1*LILRB4-STAR-T cells were added to the target cells at an effector-to-target ratio of 0.3:1. The co-culture volume was 1 mL. After co-culturing for 24 hours, the co-cultured cell suspension was collected, and the LUC luminescence value was detected using a luciferase reporter gene assay kit. The killing efficiency of STAR-T cells against target cells was calculated, as shown in Figure 20. The results showed that, with the same antibody conjugation method but different co-stimulatory domain conjugation methods, the killing abilities of NCLL05(b)-NLB14(a)STAR-abOX40 and NCLL05(b)-NLB14(a)STAR-ab(G4S)3OX40 cells, with both intracellular regions of STAR simultaneously conjugated to the OX40 co-stimulatory domain, were basically equivalent, both significantly better than the killing ability of NCLL05(b)-NLB14(a)STAR-a(G4S)3OX40. Simultaneous conjugation of OX40 to the α and β chains of CLL1*LILRB4-STAR enhanced the short-term killing ability of STAR-T cells.

[0406] 12.2 Continuous killing efficiency of CLL1*LILRB4-STAR-T with different OX40-linked structures against different target cells

[0407] MV4-11-LUC target cells expressing CLL1 and LILRB4 targets were seeded in 12-well plates at a density of 1E6 / well. As shown in Figures 19(A)-19(C), different OX40-connected CLL1*LILRB4-STAR-T cells were added to the target cells at an effector-to-target ratio of 0.5:1 (STAR-positive T cells to the target cells). The co-culture volume was 1.8 mL. Target cell killing was assessed every 48 hours. When complete target cell killing was detected, 1E6 MV4-11-LUC target cells were added to each well for continued co-culture. Target cell killing was assessed every 48 hours until no target cell killing occurred, at which point the experiment ended. As shown in Figure 21, after the time interval, the co-cultured cell suspension was collected, and the LUC luminescence value was detected using a luciferase reporter gene assay kit to calculate the killing efficiency of STAR-T cells against target cells.

[0408] Figure 21 shows the continuous killing results. The results indicate that the simultaneous connection of the α and β chains of CLL1*LILRB4-STAR to OX40 enhances the continuous killing capability of STAR-T.

Claims

1. A nanobody or antigen-binding fragment of CLL1, characterized in that, The nanobody or its antigen-binding fragment includes a heavy chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3, wherein: i) The CDR1 amino acid sequence comprises the amino acid sequence shown in SEQ ID NO 2 or contains an amino acid sequence having three, two, or one amino acid substitutions as shown in SEQ ID NO: 2; the CDR2 amino acid sequence comprises the amino acid sequence shown in SEQ ID NO: 5 or contains an amino acid sequence having three, two, or one amino acid substitutions as shown in SEQ ID NO: 5; and the CDR3 amino acid sequence comprises the amino acid sequence shown in SEQ ID NO: 8 or contains an amino acid sequence having three, two, or one amino acid substitutions as shown in SEQ ID NO:

8. ii) The CDR1 amino acid sequence comprises or contains an amino acid sequence having three, two, or one amino acid substitutions as shown in SEQ ID NO: 1; the CDR2 amino acid sequence comprises or contains an amino acid sequence having three, two, or one amino acid substitutions as shown in SEQ ID NO: 4; and the CDR3 amino acid sequence comprises or contains an amino acid sequence having three, two, or one amino acid substitutions as shown in SEQ ID NO: 7; or iii) The CDR1 amino acid sequence comprises the amino acid sequence shown in SEQ ID NO: 3 or contains an amino acid sequence having three, two, or one amino acid substitutions as shown in SEQ ID NO: 3; the CDR2 amino acid sequence comprises the amino acid sequence shown in SEQ ID NO: 6 or contains an amino acid sequence having three, two, or one amino acid substitutions as shown in SEQ ID NO: 6; and the CDR3 amino acid sequence comprises the amino acid sequence shown in SEQ ID NO: 9 or contains an amino acid sequence having three, two, or one amino acid substitutions as shown in SEQ ID NO:

9.

2. The nanobody or its antigen-binding fragment according to claim 1, characterized in that, The nanobody or its antigen-binding fragment further includes an FR region, preferably FR1-FR4 containing a heavy chain variable region; Preferably, FR1-FR4 are each independently derived from humans or non-human animals.

3. The nanobody or its antigen-binding fragment according to claim 2, characterized in that, The nanobody or its antigen-binding fragment includes FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 sequentially from the N-terminus to the C-terminus.

4. The nanobody or its antigen-binding fragment according to claim 2 or 3, characterized in that, The FR1 contains any of the amino acid sequences described in SEQ ID NO: 16-18; the FR2 contains any of the amino acid sequences described in SEQ ID NO: 19-20; the FR3 contains any of the amino acid sequences described in SEQ ID NO: 21-23; and the FR4 contains the amino acid sequence described in SEQ ID NO:

24.

5. The nanobody or its antigen-binding fragment according to any one of claims 1-4, characterized in that, The nanobody or its antigen-binding fragment contains the amino acid sequence shown in SEQ ID NO: 25, 26 or 27, or contains an amino acid sequence that has more than 70% homology with the amino acid sequence shown in SEQ ID NO: 25, 26 or 27.

6. A synthetic T-cell receptor antigen receptor (STAR), characterized in that, The STAR comprises a first peptide chain and a second peptide chain: i) The first peptide chain includes a first target-binding region and a first constant region, and the second peptide chain includes a second target-binding region and a second constant region; or... ii) The first peptide chain includes a first constant region, the second peptide chain includes a second constant region, and the first peptide chain or the second peptide chain includes a first target binding region; Wherein the first target binding region and / or the second target binding region comprises one or more antigen binding regions, and the plurality of antigen binding regions may be the same or different or partially the same; At least one of the antigen-binding regions in the first target-binding region and / or the second target-binding region contains a nanobody or antigen-binding fragment of the CLL1-binding nanobody as described in any one of claims 1-5.

7. The STAR according to claim 6, characterized in that, in, The antigen-binding region in the first target binding region and / or the second target binding region further includes an antibody or its antigen-binding fragment that specifically binds to LILRB4, EGLN3, CD33, SIGLEC-6, CD7, CD123, CD47, CD70, CD55, CD117 or CD82. Preferably, the antibody or its antigen-binding fragment is selected from scFv or nanobodies.

8. The STAR according to claim 6 or 7, characterized in that, The STAR mentioned includes any one of the following groups: a) The first peptide chain includes a first TCR constant region; the second peptide chain includes, from the N-terminus to the C-terminus, at least one nanobody or antigen-binding fragment of any one of claims 1-5, and a second TCR constant region; b) The first peptide chain comprises, from the N-terminus to the C-terminus, any of the nanobodies or antigen-binding fragments of claims 1-5, and a first TCR constant region; the second peptide chain comprises, from the N-terminus to the C-terminus, an antibody that binds to LILRB4 and a second TCR constant region. c) The first peptide chain contains a first TCR constant region; the second peptide chain contains, from the N-terminus to the C-terminus, any of the nanobodies or antigen-binding fragments of claims 1-5, an antibody that binds to LILRB4, and a second TCR constant region. d) The first peptide chain contains a first TCR constant region; the second peptide chain contains, from the N-terminus to the C-terminus, an antibody binding to LILRB4, the nanobody or its antigen-binding fragment as described in any one of claims 1-5, and a second TCR constant region.

9. The STAR according to claim 8, characterized in that, The first TCR constant region of the first peptide chain is a constant region of the TCRα chain or a variant thereof, or a constant region of the TCRβ chain or a variant thereof; the second TCR constant region of the second peptide chain is a constant region of the TCRβ chain or a variant thereof, or a constant region of the TCRα chain or a variant thereof; the TCR constant regions of the first peptide chain and the second peptide chain are not simultaneously constant regions of the TCRα chain or a variant thereof, or are not simultaneously constant regions of the TCRβ chain or a variant thereof; or The first TCR constant region of the first peptide chain is a constant region of the TCRγ chain or a variant thereof, or a constant region of the TCRδ chain or a variant thereof, and the second TCR constant region of the second peptide chain is a constant region of the TCRδ chain or a variant thereof, or a constant region of the TCRγ chain or a variant thereof; the TCR constant regions of the first peptide chain and the second peptide chain are not simultaneously constant regions of the TCRγ chain or a variant thereof, or are not simultaneously constant regions of the TCRδ chain or a variant thereof.

10. The STAR according to claim 9, characterized in that, The constant region of the TCRα chain is the constant region of the human or mouse TCRα chain; The constant region of the TCRβ chain is the constant region of the human or mouse TCRβ chain; The constant region of the TCRγ chain is the constant region of the human or mouse TCRγ chain; The constant region of the TCRδ chain is the constant region of the human or mouse TCRδ chain.

11. The STAR according to claim 9, characterized in that, The constant region variants include one or more mutations from the following group: i) N-terminal modification; ii) cysteine ​​substitution; iii) hydrophobic amino acid substitution.

12. The STAR according to claim 9, characterized in that, The native intracellular regions of the first TCR constant region and / or the second TCR constant region are deleted and / or the constant regions are humanized or mouse-derived.

13. The STAR according to any one of claims 9-12, characterized in that, 1) The sequence of the constant region of the TCRα chain or a variant thereof includes any one of SEQ ID NO: 28, 30, 106-113; and / or, the sequence of the constant region of the TCRβ chain or a variant thereof includes any one of SEQ ID NO: 29, 31, 114-119; or 2) The sequence of the constant region of the TCRγ chain or a variant thereof includes SEQ ID NO: 32 or 104; and / or, the sequence of the constant region of the TCRδ chain or a variant thereof includes SEQ ID NO: 33 or 105.

14. The STAR according to any one of claims 6-13, characterized in that, The first peptide chain and / or the second peptide chain have at least one exogenous intracellular functional domain attached to their C-terminus; Preferably, the exogenous intracellular functional domain is connected to the C-terminus of the constant region of the first peptide chain and / or the second peptide chain that is missing from the intracellular region; Preferably, the functional domain includes one or more of the following: co-stimulatory molecular fragments, co-inhibitory molecular fragments, cytokine receptor fragments, or immune cell regulatory factor fragments. Preferably, the co-stimulatory molecule includes one or more of 2B4, DAP10, DAP12, OX40, CD40, ICOS, CD28, 4-1BB, CD27, or NKp80; more preferably, the co-stimulatory molecule includes OX40; even more preferably, the intracellular domain of OX40 contains an amino acid sequence such as SEQ ID NO:

125. Preferably, the co-inhibitory molecules include one or more of TIM3, PD1, CTLA4, or LAG3; Preferably, the cytokine receptors include one or more of the following: interleukin receptors, interferon receptors, tumor necrosis factor superfamily receptors, colony-stimulating factor receptors, chemokine receptors, or growth factor receptors, such as one or more of IL-2β, IL-7α, or IL-21. Preferably, the immune cell regulatory factor is a T cell regulatory factor, such as the NIK domain; Preferably, the functional domain is a fusion protein of a cytokine receptor and a human STAT5 activation module, such as a fusion protein of IL-2β and a human STAT5 activation module or a fusion protein of IL-7α and a human STAT5 activation module.

15. The STAR according to claim 14, characterized in that, A) The first peptide chain contains, from N-terminus to C-terminus, a first TCR constant region and at least one functional domain; the second peptide chain contains, from N-terminus to C-terminus, at least one nanobody or antigen-binding fragment of any one of claims 1-5 and a second TCR constant region. B) The first peptide chain comprises, from the N-terminus to the C-terminus, any of the nanobodies or antigen-binding fragments of claims 1-5, and a first TCR constant region; the second peptide chain comprises, from the N-terminus to the C-terminus, an antibody that binds to LILRB4, a second TCR constant region, and at least one functional domain. C) The first peptide chain comprises, from N-terminus to C-terminus, a first TCR constant region and at least one functional domain; the second peptide chain comprises, from N-terminus to C-terminus, any of the nanobodies or antigen-binding fragments of claims 1-5, an antibody that binds to LILRB4, and a second TCR constant region. D) The first peptide chain contains, from the N-terminus to the C-terminus, a first TCR constant region and at least one functional domain; the second peptide chain contains, from the N-terminus to the C-terminus, an antibody that binds to LILRB4, the nanobody or its antigen-binding fragment as described in any of claims 1-5, and a second TCR constant region. E) The first peptide chain comprises, from the N-terminus to the C-terminus, any of the nanobody or antigen-binding fragments of claims 1-5, a first TCR constant region, and at least one functional domain; the second peptide chain comprises, from the N-terminus to the C-terminus, an antibody binding to LILRB4, a second TCR constant region, and at least one functional domain.

16. The STAR according to any one of claims 7-15, characterized in that, The antibody that binds to LILRB4 is a nanobody comprising CDR1 shown in SEQ ID NO: 10, CDR2 shown in SEQ ID NO: 11, and CDR3 shown in SEQ ID NO: 12; Or including nanobodies of CDR1 shown in SEQ ID NO: 13, CDR2 shown in SEQ ID NO: 14, and CDR3 shown in SEQ ID NO: 15; Preferably, the antibody binding to LILRB4 comprises a VHH nanobody as shown in SEQ ID NO: 120 or 121.

17. The STAR according to any one of claims 6-16, characterized in that, The domains in the STAR (e.g., between functional domains and the C-terminus of the constant region, between functional domains tandemly on the same peptide chain, between antigen-binding regions and the constant region, or between antigen-binding regions tandemly on the same peptide chain) are directly connected by peptide bonds or by adapters. Preferably, the connector is a (G4S)n connector or a (EAAAK)n connector, where n represents an integer from 1 to 10; Preferably, the connector includes a rigid connector, a flexible connector, a pyrolytic connector, or a meaningless amino acid; Preferably, the amino acid sequence of the rigid connector includes one or more of SEQ ID NO: 34-44; the amino acid sequence of the flexible connector includes one or more of SEQ ID NO: 45-97 or 103; and the cleavable connector includes one or more of SEQ ID NO: 98-102.

18. The STAR according to any one of claims 6-17, characterized in that, The first peptide chain of STAR is selected from SEQ ID No: 143, 145, 147, 149, 151, 153, 155, 157, 159, 161 or 167; and / or The amino acid sequence of the second peptide chain is selected from 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 168, 169 or 170.

19. A STAR complex, characterized in that, The STAR complex comprises the STAR as described in any one of claims 6-18, and one or more of CD3ε, CD3γ, CD3δ and CD3ζ; Preferably, the amino acid sequence of CD3γ includes SEQ ID NO: 138; Preferably, the amino acid sequence of CD3δ includes SEQ ID NO: 139; Preferably, the amino acid sequence of CD3ε includes SEQ ID NO: 140; Preferably, the amino acid sequence of CD3ζ includes SEQ ID NO:

136.

20. An antigen receptor, characterized in that, The antigen receptor includes the CLL1-binding nanobody or its antigen-binding fragment as described in any one of claims 1-5, or the STAR as described in claims 6-18.

21. The antigen receptor according to claim 20, characterized in that, The antigen receptor is selected from TCR, synthetic T cell receptor antigen receptor STAR, chimeric antigen receptor CAR, antibody containing a constant region, or antibody containing an Fc region.

22. A nucleic acid, characterized in that, The nucleic acid encodes the nanobody or its antigen-binding fragment as described in any one of claims 1-5, the STAR as described in claims 6-18, the STAR complex as described in claim 19, or the antigen receptor as described in any one of claims 20-21.

23. A carrier, characterized in that, The carrier comprises the nucleic acid as described in claim 22; Preferably, the expression vector comprises, from the 5' end to the 3' end, the following: The nucleotide sequence encoding the first peptide chain, the nucleotide sequence encoding the linker peptide, and the nucleotide sequence encoding the second peptide chain; or, The nucleotide sequence encoding the second peptide chain, the nucleotide sequence encoding the linker peptide, and the nucleotide sequence encoding the first peptide chain; Preferably, the linker peptide is a cleavable linker, including a 2A peptide, such as Furin-P2A; more preferably, the amino acid sequence of Furin-P2A includes SEQ ID NO:

137.

24. A host cell, characterized in that, The host cell comprises the nucleic acid of claim 22 or the vector of claim 23.

25. An immune cell, characterized in that, The immune cells express any of the nanobodies or antigen-binding fragments of claims 1-5, any of the STARs of claims 6-18, the STAR complex of claim 19, or the antigen receptor of claims 20-21.

26. The immune cell according to claim 25, characterized in that, The immune cells mentioned include one or more of the following: lymphocytes, dendritic cells, peripheral blood mononuclear cells, macrophages, granulocytes, or mast cells. Preferably, the lymphocytes are selected from T cells, TIL cells, NK cells, NKT cells, or B cells.

27. A method for preparing immune cells according to any one of claims 25-26, characterized in that, The preparation method includes transfecting the nucleic acid of claim 22 into immune cells for expression.

28. The use of a nanobody or antigen-binding fragment thereof according to any one of claims 1-5, a STAR according to any one of claims 6-18, a STAR complex according to claim 19, an antigen receptor according to any one of claims 20-21, a nucleic acid according to claim 22, a carrier according to claim 23, a host cell according to claim 24, or an immune cell according to claim 25 or 26 in the preparation of products for the diagnosis and / or treatment of tumors.

29. The application according to claim 28, characterized in that, The tumor in question is one in which reducing CLL1 expression is beneficial for treatment.

30. The application according to claim 28, characterized in that, The tumors include lymphoma, non-small cell lung cancer, leukemia, ovarian cancer, nasopharyngeal carcinoma, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma; the lymphomas are selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenström macroglobulinemia; the sarcomas are selected from osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma; Preferably, the tumor is leukemia or lymphoma; Preferably, the leukemia is selected from acute lymphoblastic leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myeloid leukemia.

31. A pharmaceutical composition or diagnostic kit, characterized in that, The pharmaceutical composition or diagnostic kit comprises the nanobody or its antigen-binding fragment as described in any one of claims 1-5, the STAR as described in any one of claims 6-18, the STAR complex as described in claim 19, the antigen receptor as described in any one of claims 20-21, the nucleic acid as described in claim 22, the carrier as described in claim 23, the host cell as described in claim 24, or the immune cell as described in claim 25 or 26.

32. A method for preventing and / or treating tumors, characterized in that, The method comprises administering to an individual in need an effective amount of any of the nanobodies or antigen-binding fragments of claims 1-5, any of the STARs of claims 6-18, the STAR complex of claim 19, the antigen receptor of claim 20 or 21, the nucleic acid of claim 22, the carrier of claim 23, the host cell of claim 24, or the immune cell of claim 25, or the pharmaceutical composition of claim 31.

33. The method according to claim 32, characterized in that, The tumor in question is one in which reducing CLL1 expression is beneficial for treatment.

34. The method according to claim 33, characterized in that, The tumors include lymphoma, non-small cell lung cancer, leukemia, ovarian cancer, nasopharyngeal carcinoma, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma; the lymphomas are selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenström macroglobulinemia; the sarcomas are selected from osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma; Preferably, the tumor is leukemia or lymphoma; Preferably, the leukemia is selected from acute lymphoblastic leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myeloid leukemia.

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