Multispecific antibody against LFA-1 signals and use thereof

Abstract

A multispecific antibody against LFA-1 signals and the use thereof. First, provided is a multispecific antibody or an antigen-binding fragment thereof, wherein the multispecific antibody comprises at least two domains, one domain (or referred to as a first domain) targets a related antigen or personalized surface antigen expressed on a tumor cell, and the other domain (or referred to as a second domain) targets LFA-1. The multispecific antibody or the antigen-binding fragment thereof can bring tumor cells and T cells and NK cells into close proximity to enhance interactions thereof, thus enhancing the tumor killing effect of immune cells.

Description

Multispecific antibodies against lfa-1 signals and uses thereof TECHNICAL FIELD

[0001] The present application relates to multispecific antibodies and uses thereof, and more particularly to bispecific antibodies against a combination of co-stimulatory signals and tumor antigens and uses thereof in treating diseases such as tumors. BACKGROUND

[0002] Cancer immunotherapy is a treatment method that uses the body's immune system to recognize and attack cancer cells. This includes various strategies such as immune checkpoint inhibitors, cancer vaccines, immune cell therapy, and specific antibody therapy.

[0003] CD28 agonists are drugs that can be used for cancer immunotherapy. CD28 is a target often considered in the process of tumor immunotherapy. CD28 is a co-stimulatory molecule on the surface of T cells that is essential for T cell activation and survival. It is also expressed in various myeloid-derived immune cells. CD28 acts as a co-stimulatory molecule to activate T cells. In the case of natural immunity, CD28 binds to CD80 protein on the surface of antigen-presenting cells (APCs) during T cell activation, providing the necessary co-stimulatory signal for T cell activation (Kathryn M Cappell, Nat Rev Clin Oncol, 2021). CD28 agonist immunotherapy aims to enhance the response capacity of T cells by activating CD28 molecules, improving their recognition and killing ability of cancer cells. This approach shows potential in enhancing anti-tumor immune responses. However, CD28 agonists can cause serious immune-related side effects, especially when over-activated, which can trigger a cytokine storm, a potentially life-threatening immune overreaction. In addition, the effects of CD28 agonists are not limited to anti-tumor T cells, as they can also activate other types of T cells, including those that can cause autoimmune reactions. Therefore, CD28 agonist immunotherapy poses safety risks and challenges in selectivity.

[0004] BiTEs (Bispecific T-Cell Engagers) is a kind of bispecific antibody (Bi-specific antibody) for tumor immunotherapy, designed to bridge T cells and cancer cells, so as to activate the killing effect of T cells on cancer cells. One end of BiTEs is usually specifically combined with T cell surface molecule CD3, and the other end is combined with a specific antigen on the surface of cancer cells, such as CD19, etc. This structure makes BiTEs can effectively guide T cells to the vicinity of cancer cells, promote the formation of immune synapses, and activate T cells to kill cancer cells. However, due to the design of targeting CD3 in BiTEs, the wide expression and strong activation signal of CD3 can cause excessive activation of T cells, non-specific killing of normal tissues, and adverse reactions such as cytokine release syndrome (Cytokine Release Syndrome, CRS); and the design of BiTEs usually depends on known antigens, and some cancers may not express enough target antigens, so the applicability of BiTEs is limited and not suitable for all types of cancer; in addition, BiTEs usually have a relatively short half-life, which may require patients to receive frequent treatment, increasing the complexity and inconvenience of treatment; some patients may also develop immune tolerance to BiTEs, reducing their therapeutic effect.

[0005] Existing tumor immunotherapy still faces a series of challenges, including fluctuations in treatment effect and different immune responses of patients, etc. There is still a need in the field to develop more drugs and safe and effective therapies to combat tumors. SUMMARY

[0006] One object of the present application is to provide a multispecific antibody that can be used to treat diseases such as tumors.

[0007] The present inventors have found in their research that a cell adhesion molecule, ICAM-1 (Intercellular Adhesion Molecule 1), plays a key role in the interaction between cytotoxic T lymphocytes (Cytotoxic T Lymphocytes, CTL) and natural killer (NK) cells (Natural Killer Cells) and tumor cells.

[0008] ICAM-1 is a surface adhesion molecule that mainly promotes intercellular adhesion by binding to lymphocyte function-associated antigen-1 (LFA-1).

[0009] CTLs exert their killing effect by recognizing specific antigens on the surface of tumor cells, a process that largely depends on the formation of an immunological synapse. The immunological synapse is a specialized intercellular contact region that serves to transmit activation signals and killing molecules. In the immunological synapse, CTLs bind to the MHC-antigen complex on the surface of tumor cells through T cell receptors (TCRs), triggering intracellular signals that activate CTLs. Subsequently, CTLs induce tumor cell apoptosis directly by releasing killing molecules such as perforin and granzyme B. In addition, the formation of the immunological synapse helps to improve the specific recognition and killing efficiency of CTLs on target cells.

[0010] NK cells play an early defense role in the immune system, capable of recognizing and killing cells that do not express or express low levels of MHC class I molecules, which is often a characteristic of tumor cells. NK cells kill tumor cells through two main mechanisms: one is to recognize stress-induced molecules on the surface of tumor cells, such as NKG2D ligands, to activate NK cells and induce cytotoxicity; the second is to release perforin and granzyme, directly causing tumor cell membrane damage and apoptosis. In addition, NK cells can also release cytokines such as interferon-γ (IFN-γ), further activating the immune response and enhancing immune surveillance of tumors.

[0011] The inventors' research shows that the interaction of ICAM-1 and LFA-1 helps to stabilize the contact between CTLs or NK cells and tumor cells, thereby promoting effective signal transmission and concentrated release of killing molecules. Tumor cells lacking expression of ICAM-1 are significantly less sensitive to killing by CTLs and NK cells. In many tumor cells, the lack or reduction of ICAM-1 expression affects the ability of CTLs and NK cells to recognize and kill tumor cells. This limits the effectiveness of existing immunotherapy on ICAM-1 deficient or low expressing tumor cells. The present invention provides a new immunotherapy that can restore the ICAM-1 / LFA-1 signal to enhance CTL and NK cell-mediated tumor killing.

[0012] According to one aspect of the present application, the present application utilizes the ICAM-1 and LFA-1 signaling axis in the co-stimulatory signal to enhance the immune system's response to tumors, and designs an innovative multi-specific antibody comprising at least two domains, wherein one domain (or first domain) targets a relevant antigen or personalized surface antigen expressed on tumor cells, and the other domain (or second domain) targets LFA-1, thereby making up for the lack of the second co-stimulatory signal due to the silence of tumor ICAM1 expression. The multi-specific antibody of the present application can bring the tumor cells and T cells and NK cells closer through the two targets, and enhance the tumor killing effect of immune cells. This design of the present application is not only expected to improve the therapeutic effect of existing immunotherapy, but also can cope with the strategy of immune escape of tumors, and bring a more comprehensive solution to the field of cancer treatment.

[0013] According to a specific embodiment of the present application, the present application provides a multi-specific antibody or an antigen-binding fragment thereof, comprising at least two domains, wherein one domain targets a relevant antigen or personalized surface antigen expressed on tumor cells, and the other domain targets LFA-1.

[0014] According to a specific embodiment of the present application, the multi-specific antibody or an antigen-binding fragment thereof of the present application, wherein the tumor-related antigen includes but is not limited to one or more of the following: an antigen of a relevant tumor expressing EGFR, an antigen of a relevant tumor expressing HER2, an antigen of a relevant tumor expressing PDL1, an antigen of a relevant tumor expressing CD19, an antigen of a relevant tumor expressing CD20, an antigen of a relevant tumor expressing CLL1, an antigen of a relevant tumor expressing CD22, an antigen of a relevant tumor expressing CD30, an antigen of a relevant tumor expressing BCMA, an antigen of a relevant tumor expressing EGFRvIII, an antigen of a relevant tumor expressing PSMA, an antigen of a relevant tumor expressing Muc1, an antigen of a relevant tumor expressing Claudin7, an antigen of a relevant tumor expressing TSA, an antigen of a relevant tumor expressing MSLN, an antigen of a relevant tumor expressing GPC3, an antigen of a relevant tumor expressing IL13RA2, an antigen of a relevant tumor expressing SLAMF7, an antigen of a relevant tumor expressing GPRC5D, an antigen of a relevant tumor expressing LILRB4, an antigen of a relevant tumor expressing DLL3, an antigen of a relevant tumor expressing TROP2, and an antigen of a relevant tumor expressing Claudin18.2.

[0015] According to a specific embodiment of the present application, the multi-specific antibody or an antigen-binding fragment thereof of the present application, wherein:

[0016] Tumors associated with expression of EGFR include, but are not limited to, colorectal cancer, renal cancer, non-small cell lung cancer, pancreatic cancer, nasopharyngeal cancer, head and neck cancer, etc.

[0017] Tumors associated with expression of HER2 include, but are not limited to, breast cancer, ovarian cancer, endometrial cancer, fallopian tube cancer, gastric cancer, and prostate cancer, etc.

[0018] Tumors associated with expression of CD19 include, but are not limited to, acute lymphoblastic leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, etc.

[0019] Tumors associated with expression of CD20 include, but are not limited to, acute lymphoblastic leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, etc.

[0020] Tumors associated with expression of Claudin 18.2 include, but are not limited to, gastric cancer / gastroesophageal junction cancer, breast cancer, colon cancer, head and neck cancer, bronchial cancer, and non-small cell lung cancer, etc.

[0021] According to a specific embodiment of the present application, the multispecific antibody or antigen-binding fragment thereof of the present application, wherein the tumor-associated antigen is selected from one or more of CLL1, CD19, CD20, CD22, CD30, BCMA, EGFR, EGFRvIII, PSMA, Muc1, Claudin7, TSA, MSLN, GPC3, IL13RA2, SLAMF7, GPRC5D, LILRB4, DLL3, TROP2, PDL1, Claudin6, B7-H3, FAP, CD123, Claudin18.2.

[0022] According to a specific embodiment of the present application, the multispecific antibody or antigen-binding fragment thereof of the present application, wherein the tumor-associated antigen is from a tumor of epithelial origin expressing EGFR or a tumor expressing HER2.

[0023] According to a specific embodiment of the present application, the multispecific antibody or antigen-binding fragment thereof of the present application, wherein the domain targeting a tumor-associated antigen is from an amino acid sequence of an antibody or antigen-binding fragment thereof that can bind to the tumor-associated antigen.

[0024] According to a specific embodiment of the present application, the multispecific antibody or antigen-binding fragment thereof of the present application, wherein the domain targeting a tumor-associated antigen is from an anti-EGFR antibody.

[0025] According to a specific embodiment of the application, the multispecific antibody or antigen binding fragment thereof of the application, wherein the domain targeting a tumor associated antigen comprises a first heavy chain variable region VH1 comprising the amino acid sequences of HCDR 1-3 as set forth in SEQ ID NO. 1-3, respectively; and a first light chain variable region VL1 comprising the amino acid sequences of LCDR 1-3 as set forth in SEQ ID NO. 4-6, respectively:

[0026] HCDR 1: NYGVH (SEQ ID NO. 1)

[0027] HCDR 2: VIWSGGNTDYNTPFTS (SEQ ID NO. 2)

[0028] HCDR 3: ALTYYDYEFAY (SEQ ID NO. 3)

[0029] LCDR 1: RASQSIGTNIH (SEQ ID NO. 4)

[0030] LCDR 2: YASESIS (SEQ ID NO. 5)

[0031] LCDR 3: QQNNNWPTT (SEQ ID NO. 6).

[0032] According to a specific embodiment of the application, the multispecific antibody or antigen binding fragment thereof of the application, wherein the domain targeting a tumor associated antigen comprises a first heavy chain variable region VH1 comprising the amino acid sequences of HCDR 1-3 as set forth in SEQ ID NO. 1-3, respectively; and a first light chain variable region VL1 comprising the amino acid sequences of LCDR 1-3 as set forth in SEQ ID NO. 4-6, respectively:

[0033] the first heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% sequence identity to SEQ ID NO. 7:

[0034] the first light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO. 8:

[0035] According to a specific embodiment of the application, the multispecific antibody or antigen binding fragment thereof of the application, wherein the domain targeting a tumor associated antigen comprises a scFv fragment.

[0036] According to a specific embodiment of the application, the multispecific antibody or antigen binding fragment thereof of the application, wherein the domain targeting a tumor associated antigen comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO. 9:

[0037] According to a specific embodiment of the application, the multispecific antibody or antigen binding fragment thereof of the application, wherein the domain targeting a tumor associated antigen comprises an amino acid sequence of Cetuximab or an antigen binding fragment thereof.

[0038] According to a specific embodiment of the application, the multispecific antibody or antigen binding fragment thereof of the application, wherein the domain targeting LFA-1 has a sequence from a LFA-1 binding protein. The LFA-1 binding protein can be any protein available in the art that can bind to LFA-1, for example, see the article by Hongmin Zhang et al. 2009, "Structural basis of activation-dependent binding of ligand-mimetic antibody AL-57 to integrin LFA-1" (Proc Natl Acad Sci U S A. 2009 Oct 27; 106(43): 18345-50. doi: 10.1073 / pnas.0909301106. Epub 2009 Sep 23.) describing the AL-57 antibody as a mimetic antibody that activates the activated LFA-1, PUBMED sequence: 19805116. The LFA-1 binding protein can bind to different conformations of LFA-1 depending on the spatial conformation of LFA-1, but not necessarily have the function of activating or inhibiting the LFA-1 signal.

[0039] According to a specific embodiment of the application, the multispecific antibody or antigen binding fragment thereof of the application, wherein the LFA-1 binding protein is a LFA-1 agonistic protein.

[0040] According to a specific embodiment of the application, the multispecific antibody or antigen binding fragment thereof of the application, wherein the LFA-1 binding protein is an antibody that can bind to LFA-1.

[0041] According to a specific embodiment of the application, the multispecific antibody or antigen binding fragment thereof of the application, wherein the LFA-1 binding protein is selected from one or more of the following proteins:

[0042] AL57 (e.g., CmScFvXAL-57), CBR LFA-1 / 2, CBR LFA-1 / 7, MEM83, TS2 / 4; these antibodies are commercially available from T. A. Springer (Boston Children’s Hospital, Boston, MA);

[0043] 7E4, R2E7B, 17MEM48, MEM148, anti-phospho-b1 Thr-788 / 789, 12G10-488; these antibodies are commercially available from Abeam (Cambridge, United Kingdom);

[0044] TS1 / 22, TS1 / 18, anti-a4; these antibodies are commercially available from Thermo Scientific (Waltham, MA);

[0045] MHM23; this antibody is commercially available from Dako (Glostrup, Denmark);

[0046] MHM24; this antibody is commercially available from DSHB (Iowa City, IA);

[0047] Cet x ICAM1-D1.

[0048] According to a specific embodiment of the present application, the multispecific antibody or antigen binding fragment thereof of the present application, wherein the domain sequence targeting LFA-1 is from LFA-1 agonistic protein or from D1 domain of ICAM-1.

[0049] According to a specific embodiment of the present application, the multispecific antibody or antigen binding fragment thereof of the present application, wherein the domain sequence targeting LFA-1 is from LFA-1 agonistic antibody, such as AL-57 or AL-57 variants, or single chain antibody (ScFv) sequence of AL57 can also be selected as sequence design according to antibody design requirements.

[0050] According to a specific embodiment of the present application, the multispecific antibody or antigen binding fragment thereof of the present application, wherein the domain sequence targeting LFA-1 comprises a second heavy chain variable region VH2 and a second light chain variable region VL2, the VH2 comprises CDR sequences of heavy chain as shown in SEQ ID NO. 10, respectively; the VL2 comprises CDR sequences of light chain as shown in SEQ ID NO. 11, respectively:

[0051] According to a specific embodiment of the present application, the multispecific antibody or antigen binding fragment thereof of the present application, wherein,

[0052] said second heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% sequence identity with SEQ ID NO. 10;

[0053] said second light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity with SEQ ID NO. 11.

[0054] According to a specific embodiment of the application, the multispecific antibody or antigen binding fragment thereof of the application, wherein said domain sequence targeting LFA-1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO. 12:

[0055] According to a specific embodiment of the application, the multispecific antibody or antigen binding fragment thereof of the application, comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.4%, at least 99.6%, at least 99.7%, at least 99.8%, or 100% sequence identity with SEQ ID NO. 13:

[0056] In some embodiments of the application, the sequence of ICAM-1 mimicking ligand AL-57 is used as a domain targeting LFA-1 (US20110212112A1). By mimicking the binding of the tumor costimulatory molecule ICAM-1 to its ligand LFA-1, the absence of which on the tumor cell surface is complemented, the interaction of immune cells with tumor cells is enhanced.

[0057] According to a specific embodiment of the application, the multispecific antibody or antigen binding fragment thereof of the application, wherein said personalized surface antigen is, for example, the epithelial cell marker EpCAM or the like.

[0058] In some embodiments of the application, said multispecific antibody is a bispecific antibody.

[0059] According to a specific embodiment of the application, the multispecific antibody or antigen binding fragment thereof of the application is a bispecific antibody targeting LFA-1 and EGFR.

[0060] According to a specific embodiment of the present application, the bispecific antibody or antigen-binding fragment thereof of the present application is provided as shown in the schematic of Figure 9, wherein the Fab fragment is Cetuximab targeting EGFR on the surface of tumor cells and the Fc end is D1 domain of ICAM-1 targeting LFA-1 on the surface of T cells.

[0061] The multispecific antibody or antigen-binding fragment thereof of the present application includes "conservative sequence modifications" thereof, i.e. nucleotide and amino acid sequence modifications that do not significantly affect and alter the binding characteristics of the antibody or the antibody containing the amino acid sequence. The conservative sequence modifications include nucleotide or amino acid substitutions, additions or deletions. The modifications can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis, etc. Conservative amino acid substitutions include the replacement of an amino acid residue by another having similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine). Thus, preferably, another amino acid residue from the same side chain family is substituted for a non-essential amino acid residue in the human anti-TROP2 antibody. Antibodies having the disclosed amino acid sequence and / or antibodies containing the disclosed amino acid sequence disclosed above, including antibodies substantially encoded by or containing similar sequences that have been conservatively modified, are considered to be within the scope of the present application.

[0062] In another aspect, the present application also provides a nucleic acid molecule encoding the multispecific antibody or antigen-binding fragment thereof of the present application. In view of the degeneracy of the genetic code, the gene encoding the antibody of the present application can be modified in its coding region without changing the amino acid sequence, i.e. the gene sequence encoding the above-mentioned antibody is modified to obtain a gene encoding the same antibody. The modified gene can be artificially synthesized by a person skilled in the art according to the codon bias of the host expressing the antibody to improve the expression efficiency of the antibody.

[0063] In another aspect, the present application also provides an expression vector containing the nucleic acid molecule of the present application.

[0064] In another aspect, the present application also provides a recombinant cell comprising the nucleic acid molecule of the present application.

[0065] According to another aspect of the present application, the present application also provides a method for preparing the multispecific antibody or antigen binding fragment thereof of the present application.

[0066] According to a specific embodiment of the present application, the present application can construct and prepare the multispecific antibody, particularly bispecific antibody, by means of genetic engineering and protein engineering, for reestablishing the synaptic signal between tumor and immune cells. In the present application, the bispecific antibody is used to establish the synapse between tumor and immune cells, which is named as Bispecific Synapse Engager.

[0067] According to a specific embodiment of the present application, the method for preparing the multispecific antibody of the present application comprises:

[0068] constructing a plasmid comprising a nucleic acid sequence encoding the first domain and a plasmid comprising a nucleic acid sequence encoding the second domain; or, constructing a plasmid comprising a nucleic acid sequence encoding the first domain and a nucleic acid sequence encoding the second domain;

[0069] transfecting the plasmid into a host cell to produce a fusion protein comprising the first domain and the second domain, thereby obtaining the multispecific antibody.

[0070] In some specific embodiments of the present application, the present application provides a method for preparing a bispecific antibody, which comprises:

[0071] Step one: constructing plasmid. Using vector pHage-CMV, 1) concatenating the single chain variable fragment (CmScFv) of Cetuximab and the Fc variable region of Knob in one plasmid, 2) concatenating the heavy chain of AL-57 and the Fc variable region of hole in one plasmid, 3) directly constructing the light chain of AL-57 in the vector.

[0072] Step two: transfecting cells to express protein. Transiently co-transfecting the plasmid of step one into FreeStyleTM 293-F cells to produce protein, and then collecting the supernatant for protein purification. The fusion protein will form a dimer through the heterodimeric Fc variant KiHss-AkKh platform.

[0073] Step three: protein purification and verification of composition. Purifying the supernatant containing the fusion protein using protein A affinity chromatography. Then, heating and denaturing the antibody using reducing protein buffer and non-reducing protein buffer respectively, and then verifying whether the composition of the fusion protein conforms to the designed sequence by the band size of SDS-PAGE protein gel.

[0074] Step four: affinity determination of bispecific antibody. Endogenous EGFR expressing tumor cells were incubated with serially diluted antibodies for 30 minutes, followed by incubation with fluorophore conjugated anti-human IgG secondary antibody. The mean fluorescence intensity was then measured by flow cytometry, and the affinity curve was plotted.

[0075] Step five: functional verification of bispecific antibody. The function of bispecific antibody in anti-tumor immunity was verified by tumor and immune cell co-culture experiment and mouse in vivo tumor inoculation experiment, respectively.

[0076] According to another aspect of the present application, the present application also provides the use of the multispecific antibody or antigen binding fragment thereof in treating tumor. Specifically, the use includes the use of the multispecific antibody or antigen binding fragment thereof in the preparation of a medicament for treating tumor.

[0077] The present application also provides a medicament for treating tumor, which comprises an effective amount of the multispecific antibody or antigen binding fragment thereof of the present application, and a pharmaceutically acceptable carrier and / or excipient.

[0078] The present application also provides a method for treating tumor, which comprises administering to a subject an effective amount of the multispecific antibody or antigen binding fragment thereof of the present application or a medicament comprising the same.

[0079] According to a specific embodiment of the present application, in the present application, the tumor includes one or more of the following: EGFR-expressing related tumor, HER2-expressing related tumor, PD-L1-expressing related tumor, CD19-expressing related tumor, CD20-expressing related tumor, CLL1-expressing related tumor, CD22-expressing related tumor, CD30-expressing related tumor, BCMA-expressing related tumor, EGFRvIII-expressing related tumor, PSMA-expressing related tumor, Muc1-expressing related tumor, Claudin7-expressing related tumor, TSA-expressing related tumor, MSLN-expressing related tumor, GPC3-expressing related tumor, IL13RA2-expressing related tumor, SLAMF7-expressing related tumor, GPRC5D-expressing related tumor, LILRB4-expressing related tumor, DLL3-expressing related tumor, TROP2-expressing related tumor, Claudin 6-expressing related tumor, B7-H3-expressing related tumor, FAP-expressing related tumor, CD123-expressing related tumor, Claudin18.2-expressing related tumor.

[0080] According to a specific embodiment of the present application, the specific types of tumor expressing related antigens in the present application can refer to the reports in the prior art, for example:

[0081] The tumors related to EGFR expression include, but are not limited to, colorectal cancer, kidney cancer, non-small cell lung cancer, pancreatic cancer, nasopharyngeal cancer, head and neck tumor, etc.

[0082] The tumors related to HER2 expression include, but are not limited to, breast cancer, ovarian cancer, endometrial cancer, fallopian tube cancer, gastric cancer and prostate cancer, etc.

[0083] The tumors related to CD19 and CD20 expression include, but are not limited to, acute lymphoblastic leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, etc.

[0084] The tumors related to Claudin18.2 expression include, but are not limited to, gastric cancer / gastroesophageal junction cancer, breast cancer, colon cancer, head and neck cancer, bronchial cancer and non-small cell lung cancer, etc.

[0085] The technical scheme of the present application has the following beneficial effects:

[0086] (1) Enhancing the directional attack of immune cells on tumors and reducing the side effects of treatment: The multispecific antibody of the present application mainly provides a costimulatory signal after stimulation of tumor-specific antigen signals, which can more accurately target tumor cells and activate T cells and NK cells for killing. The present application can effectively avoid the non-specific activation and killing caused by the CD3 target in the prior art, and reduce the adverse symptoms such as immunotoxicity or cytokine release syndrome.

[0087] (2) New use of costimulatory signal pathway and development of new target: The multispecific antibody of the present application uses the costimulatory signal (ICAM-1 / LFA-1 axis) as the target of the bispecific antibody, which provides a costimulatory signal for T cells and NK cells, and can avoid the immune escape mediated by the absence of costimulatory signals in tumor cells, and achieve stronger and longer immune activation.

[0088] (3) Widely applicable to different tumor types: The present application is characterized by providing a costimulatory signal, and thus does not depend on a certain specific tumor antigen, and has good applicability in different types of tumors, and has wider clinical application value. BRIEF DESCRIPTION OF DRAWINGS

[0089] Figure 1 is a schematic diagram of the preparation process of the bispecific antibody of the present application.

[0090] Figure 2 is a schematic diagram of the structure of the bispecific antibody of the present application. The bispecific antibody of the present application shown in the figure has a single-chain variable fragment (CmScFv) of Cetuximab (Cetuximab) in series with a Knob Fc variable region at one end, and an ICAM-1 mimetic ligand AL-57 fragment in series with a hole Fc variable region at the other end.

[0091] Figure 3 shows the configuration of the bispecific antibody of the present application and the results of affinity assay.

[0092] Figure 4A and Figure 4B show that the bispecific antibody of the present application can activate CD8 + T cells through co-stimulatory signal. The expression of CD69 and IFNg by the cells after activation by the antibody was detected by flow cytometry. This was used as an indicator of activation of CD8 + T cells. Figure 4A is a representative flow plot. Figure 4B is a statistical analysis bar chart.

[0093] Figure 5 shows that the bispecific antibody of the present application can enhance CD8 + T cell-mediated cytotoxicity against ICAM-1 -depleted tumor cells. The tumor and CD8 + T cell co-culture system was incubated with different antibodies at the same concentration. The expression of CD69 and IFNg by the CD8 + T cells after activation by the antibody was detected by flow cytometry. This was used as an indicator of activation of CD8

[0094] Figure 6 shows that the bispecific antibody of the present application can enhance CD8 + T cell-mediated killing of ICAM-1 -depleted tumor cells. The tumor and CD8 + T cell co-culture system was incubated with different antibodies at the same concentration. The expression of CD69 and IFNg by the CD8 + T cells after activation by the antibody was detected by flow cytometry. This was used as an indicator of activation of CD8

[0095] Figure 7 shows that the bispecific antibody of the present application can enhance NK cell-mediated cytotoxicity against ICAM-1 -depleted tumor cells. The tumor and NK cell co-culture system was incubated with different antibodies at the same concentration. The expression of CD69 and IFNg by the NK cells after activation by the antibody was detected by flow cytometry. This was used as an indicator of activation of NK cells.

[0096] Figure 8 shows that the bispecific antibody of the present application can enhance NK cell-mediated killing of ICAM-1 -depleted tumor cells. The tumor and NK cell co-culture system was incubated with different antibodies at the same concentration. The expression of CD69 and IFNg by the NK cells after activation by the antibody was detected by flow cytometry. This was used as an indicator of activation of NK cells.

[0097] Figure 9 is a schematic diagram of the structure and working of the bispecific antibody of Example 6 of the present application.

[0098] Figure 10 shows the composition and affinity determination results of the bispecific antibody of the present application. The bispecific antibody of the present application LFA-1 Engager Cet x ICAM1-D1, Fab end is Cetuximab, Fc end is human IgGl with mouse or human ICAM-1 extracellular D1 domain in tandem.

[0099] Figure 11 shows the LFA-1 Engager Cet x ICAM1-D1 of the present application enhances IFN-γ expression in mouse CD8 + T cells.

[0100] Figure 12 shows the LFA-1 Engager Cet x ICAM1-D1 of the present application functions in a TCR-MHC signal dependent manner.

[0101] Figure 13 shows the LFA-1 Engager Cet x ICAM1-D1 of the present application functions in a LFA-1 molecule dependent manner.

[0102] Figure 14 shows the human version LFA-1 Engager Cet x hICAM1-D1 of the present application enhances IFN-γ expression in PBMC derived CD8+ T cells.

[0103] Figure 15 shows the anti-tumor function of the LFA-1 Engager Cet x ICAM1-D1 of the present application in MC38 and B16F10 tumor mouse models.

[0104] Figure 16 shows the LFA-1 Engager Cet x ICAM1-D1 of the present application has a synergistic anti-tumor effect with anti-PD-1.

[0105] Figure 17 shows the LFA-1 Engager Cet x ICAM1-D1 and Cetuximab treated MC38 tumor intratumoral T cell single cell transcriptome analysis of the present application.

[0106] Figure 18 shows the LFA-1 Engager Cet x ICAM1-D1 and Cetuximab treated MC38 tumor intratumoral T cell subpopulation single cell transcriptome level changes of the present application. DETAILED DESCRIPTION

[0107] Before further description of the specific embodiments of the application, it is to be understood that the application is not limited to the particular specific embodiments described; it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present application.

[0108] When a range of values is presented, unless otherwise stated, it is understood that every intervening value between the two ends, to the tenth of the unit of the lower limit unless otherwise indicated, and any other stated or intervening value in that range is encompassed. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, exemplary methods and materials are described herein.

[0109] Unless otherwise indicated, the experimental methods, detection methods, preparation methods disclosed in the present application all adopt the conventional techniques in the art.

[0110] For the purposes of the present application, certain technical and scientific terms are specifically defined below. Unless specifically defined herein, all other technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art.

[0111] In this specification, references to "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", "implementations", and the like, mean that the particular element(s) (e.g., feature, structure, property, and / or characteristic) described is included in at least one embodiment of the application described herein, and can or can not be present in other embodiments. In addition, it is to be understood that the described elements can be combined in any suitable manner in the various embodiments.

[0112] All publications and other references mentioned herein are incorporated by reference in their entirety.

[0113] antibodies, antibody fragments, and multispecific antibodies (e.g., bispecific antibodies) that contain at least two antigen binding regions. Antibodies can contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations in glycosylation sites. Antibodies also include post-translationally modified antibodies, fusion proteins containing an antigenic determinant of an antibody, and any other modified immunoglobulin molecule containing an antigen recognition site, so long as the antibodies exhibit the desired biological activity.

[0114] The term "multispecific antibody" is an engineered antibody that can bind to multiple different antigens or multiple different epitopes of an antigen simultaneously. The term "bispecific antibody" is a specific type of multispecific antibody that can bind to two different antigens or two different epitopes of an antigen simultaneously. This specificity gives them unique advantages in targeted therapies (such as for specific cancer cells) and in treating complex diseases (such as cancer or autoimmune diseases).

[0115] The term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antigen-binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fab' fragment, which is simply a Fab with part of the hinge region; (iv) a Fd fragment consisting of the VH and CH1 domains; (v) a Fd' fragment consisting of the VH and CH1 domains and one or more cysteine residues at the C-terminus of the CH1 domain; (vi) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (vii) a dAb fragment, which consists of a VH domain; (viii) an isolated complementarity determining region (CDR); and (ix) a nanobody, a heavy chain variable region containing a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv)). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Furthermore, the term also includes "linear antibodies", comprising a pair of tandem Fd segments (VH-CH1-VH-CH1), which, together with complementary light chain polypeptides, form a pair of antigen binding regions at the amino or carboxy termini of the linear antibody. These antigen binding fragments are obtained using conventional techniques known to those with ordinary skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0116] The terms "binds" or "specifically binds" refer to a non-random binding reaction between two molecules, such as an antibody and its target antigen. The binding specificity of an antibody can be determined based on affinity and / or avidity. Affinity represents the equilibrium constant for dissociation of an antigen from an antibody (KD), a measure of the strength of binding between an antigenic determinant and an antigen binding site of an antibody: the smaller the value of KD, the stronger the binding strength between the antigenic determinant and the antibody. Alternatively, affinity can also be expressed as the affinity constant (KA), which is 1 / KD. Avidity is a measure of the binding strength between an antibody and the associated antigen. Avidity involves both the affinity between the antigenic determinant and the antigen binding site of an antibody and the number of available binding sites on the antibody. Specific binding of an antibody to an antigen or antigenic determinant can be determined in any known suitable manner, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competition assays, as well as different variations thereof known in the art.

[0117] The term "epitope" refers to a site on an antigen to which an antibody binds. Epitopes can be formed both by contiguous amino acids or by amino acids that are noncontiguous in sequence as a result of protein folding to bring the amino acids into proximity. Epitopes formed from contiguous amino acids (also known as linear epitopes) are generally retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding (also known as conformational epitopes) are generally lost upon treatment with denaturing solvents. Epitopes usually include at least 3, and more usually at least 5 or 8- 10 amino acids in a unique spatial conformation. Epitopes define the minimal binding site for an antibody and are therefore the specific targets of antibodies or antigen-binding fragments thereof.

[0118] The term "sequence identity" refers to the extent to which two sequences (amino acid) have identical residues in the same positions when aligned. For example, "an amino acid sequence is X% identical to SEQ ID NO: Y" means that the amino acid sequence has X% identity to SEQ ID NO: Y and is set forth as X% of the residues in the amino acid sequence are identical to the residues of the sequence disclosed in SEQ ID NO: Y. Typically, such calculations are performed using a computer program. Exemplary programs for comparing and aligning pairs of sequences include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990), and gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984). In addition, when determining the extent of sequence identity between two amino acid sequences, the skilled artisan can take into account so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions that replace an amino acid residue with another amino acid residue having similar chemical structure that have little or substantially no effect on the function, activity, or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art. Such conservative substitutions are preferably substitutions of one amino acid for another amino acid in the same group (a) to (e) below: (a) small aliphatic, nonpolar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gin; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp. Particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gin or to His; Asp to Glu; Cys to Ser; Gin to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gin; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gin, or to Glu; Met to Leu, to Tyr, or to Ile; Phe to Met, to Leu, or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to Ile, or to Leu.

[0119] The term“tumor-associated antigen” or“relevant antigen expressed on tumor cells” refers to those antigens that are expressed on tumor cells and are differentially expressed or expressed at different levels from normal cells. These antigens can be recognized by the immune system and are sometimes used for cancer diagnosis and treatment (such as cancer vaccines or immunotherapy).

[0120] The term“immune co-stimulatory molecule” refers to molecules that play an auxiliary role in the immune response, which can enhance or modulate the activation of immune cells. These molecules are crucial for the activation of the immune system, particularly in terms of antibody production, cytotoxic T cell generation, and regulatory T cell activity.

[0121] The term“lymphocyte function-associated antigen-1” or“LFA-1” is a cell adhesion molecule found on the surface of various immune cells. The term“LFA-1 signaling pathway” refers to the interaction of LFA-1 with its ligands (such as ICAM molecules), which is critical for the migration, activation, and effector functions of immune cells.

[0122] The term“intercellular adhesion molecule 1” or“ICAM-1 molecule” is a cell surface molecule that primarily functions in regulating cell-to-cell adhesion. It is an important ligand for LFA-1, and its binding to LFA-1 is particularly important for the adhesion and migration of immune cells.

[0123] The term“vector” is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.

[0124] The term“host cell” refers to a cell into which an expression vector has been introduced.

[0125] The term“pharmaceutically acceptable” means that the carrier or excipient is compatible with the other ingredients of the composition and not deleterious or substantially deleterious to the recipient thereof, and / or such carrier or excipient is approved or approvable for inclusion in a pharmaceutical composition intended for parenteral administration to humans.

[0126] The terms "treatment," "therapy," "treat," and the like, refer to the application of an agent or performance of a procedure for the purpose of effecting an outcome. The outcomes can be prophylactic, in terms of completely or partially preventing a disease or symptom thereof, and / or therapeutic, in terms of partially or completely curing a disease and / or symptoms of a disease. As used herein, "treatment" can include treatment of a disease or condition (e.g., cancer) in a mammal, particularly in a human, and includes: (a) preventing the disease or symptom of the disease from occurring in an individual which can be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease. Treatment can refer to any successful indication in the treatment or amelioration or prevention of cancer, including any objective or subjective parameter as a consequence of, e.g., disease progression. Treatment also refers to the treatment or amelioration of disease symptoms or conditions indicated for the treatment. Treatment also includes achieving a therapeutic benefit and / or an improved prognosis. By therapeutic benefit is meant eradication or amelioration of symptoms, diminishment of scope of disease, prevention of disease, or a prejudice, or any other scientifically suitable therapeutic endpoint. Thus, the term "treatment" includes the application of an antibody or composition or conjugate disclosed herein to prevent or delay, alleviate, or ameliorate a symptom or condition associated with a disease (e.g., cancer). The term "therapeutic effect" refers to the reduction, elimination, or prevention of a disease, a symptom of a disease, or a side effect of a disease in a subject.

[0127] The term "effective amount" refers to an amount of a drug to be administered to a subject to treat a disease sufficient to effect treatment of the disease.

[0128] The term "subject" refers to any mammalian subject or human for whom diagnosis, treatment, or therapy is desired. A "mammal" for treatment purposes refers to any animal classified as a mammal, including humans, domestic and farm animals, and laboratory and sport or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, and the like.

[0129] Example 1, Preparation and detection of bispecific antibodies

[0130] The procedure for preparing the bispecific antibodies of the present invention (see Figure 2 for structure) includes (see Figure 1):

[0131] Step 1: Constructing plasmids. Using vector pHage-CMV, 1) Cetuximab (CmScFv) and knob Fc variable region were linked in one plasmid, 2) AL-57 heavy chain and hole Fc variable region were linked in one plasmid, 3) AL-57 light chain was directly constructed in the vector.

[0132] Step 2: Transfecting cells, expressing proteins. The plasmids from Step 1 were transiently co-transfected into FreeStyle 293F cells (Invitrogen) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.TM 293-F cells were used to produce proteins, and the supernatant was collected for protein purification. The fusion proteins will form dimers through the heterodimeric Fc variant KiHss-AkKh platform.

[0133] Step three: Protein purification and verification of the structure. The supernatant containing the fusion protein was purified using protein A affinity chromatography. The antibody was then denatured by heating using reducing and non-reducing protein buffers, respectively, and the fusion protein was then verified for the presence of the designed sequence by band size on SDS-PAGE protein gel.

[0134] Step four: Affinity determination of the bispecific antibody.

[0135] Human system: Endogenously EGFR-expressing SW480 or A498 tumor cells were incubated with serially diluted bispecific antibodies for 30 minutes. The supernatant was then removed, and the cells were washed once with PBS. Secondary antibody incubation was performed using anti-human IgG Fc-APC (Biolegend 410711) at 4°C for 20 minutes. After completion, the cells were washed once with PBS. Flow cytometry was used to analyze the APC, and the affinity curve of Cetuximab to EGFR was obtained.

[0136] Mouse system: The affinity of Cetuximab to EGFR was determined for various fusion proteins. The tumor cells used, MC38-EGFR and B16F10-EGFR, are a modified tumor cell line that expresses EGFR that can be recognized and bound by Cetuximab, while the EGFR of MC38 WT cannot be recognized and bound by Cetuximab. The tumor cells were incubated with serially diluted bispecific antibodies for 30 minutes. The supernatant was then removed, and the cells were washed once with PBS. Secondary antibody incubation was performed using anti-human IgG Fc-APC (Biolegend 410711) at 4°C for 20 minutes. After completion, the cells were washed once with PBS. Flow cytometry was used to analyze the APC, and the affinity curve of Cetuximab to EGFR was obtained.

[0137] The results of the bispecific antibody structure and affinity determination of the present application are shown in Figure 3.

[0138] Step five: Functional verification of the bispecific antibody. The function of the bispecific antibody in anti-tumor immunity was verified through tumor and immune cell co-culture experiments and mouse in vivo tumor inoculation experiments, respectively.

[0139] CmScFv x AL-57

[0140] Protein Sequence of AL57 Light Chain:

[0141] Protein Sequence of AL57 Heavy Chian:

[0142] Protein Sequence of ScFv of Cetuximab:

[0143] Cet x ICAM1-D1

[0144] Protein Sequence of Heavy chain of Cetuximab:

[0145] Protein Sequence of Light chain of Cetuximab:

[0146] Protein Sequence of Domain1 in ICAM-1:

[0147] Protein Sequence of heavy chain of Cetuximab and D1 of ICAM1:

[0148] (underlined part in sequence is CDR sequence)

[0149] Example 2, Packaging virus

[0150] The purpose plasmid of all lentivirus vectors is mixed with the packaging plasmid psPAX2 and pMD2.G in the ratio of 3:2:1 in Opti-MEM medium. Dissolved in Opti-MEM medium by mixing with PEI Max at 1:3.

[0151] The purpose plasmid of all retrovirus vectors is mixed with the packaging plasmid in the ratio of 1:1 in Opti-MEM medium. Dissolved in Opti-MEM medium by mixing with PEI Max at 1:3.

[0152] After mixing the plasmid dilution with the PEI Max dilution, let it stand for 20-30 minutes, then add to the pre-plated HEK293T cells. After 6-8 hours, replace the medium with fresh medium. Collect the virus 48 hours after transfection and filter through a 0.45 μm filter to obtain the lentivirus or retrovirus.

[0153] Example 3, Virus concentration and isolation and construction of NY-ESO-1 TCR T cells

[0154] Step 1: After collecting and filtering the HA-tagged NY-ESO-1 TCR lentivirus, add 5x PEG8000 solution, mix well and place at 4°C overnight. On the second day, centrifuge the virus at 4°C, 4000 rpm for 30 minutes. Discard the supernatant and use serum-free RPMI 1640 medium to concentrate the virus precipitate at a volume concentration of 100 times, then centrifuge at room temperature, 12000 rpm for 1 minute. Take the supernatant to obtain the lentivirus for infecting T cells.

[0155] Step 2: On the first day, thaw the PBMC.

[0156] Step 3: On the second day, use human CD8 + T cell enrichment isolation kit to isolate CD8 + T cells, and plate 0.5x10 6 CD8 + T cells in a 48-well plate pre-coated with human anti-CD3 (5 μg / mL), anti-CD28 (1 μg / mL) and human fibronectin (5 μg / mL) for activation. Then use RetroNectin (15 μg / ml) to coat a non-tissue culture 24-well plate and place at 4°C overnight.

[0157] Step 4: On the third day, discard the RetroNectin coating and block with 2% BSA at room temperature for 15 minutes. After blocking, add the concentrated lentivirus containing the HA-tagged NY-ESO-1 TCR to the coated 12-well plate, and centrifuge the culture plate at 32°C, 2000g for 2.5 hours. After centrifugation, transfer the activated T cells to the virus-coated cell culture plate and add 300 mL of human T cell culture medium, centrifuge at 600g for 5 minutes, then incubate at 37°C and 5% CO2. After three days of infection, use a flow cytometer to sort CD8 + T cells positive for IgG(H+L) labeled HA tag for subsequent experiments.

[0158] Example 4, Isolation and culture of murine T cells

[0159] Step 1: Day 1, after euthanizing OT-1 mice, remove spleen and grind, and filter through 70 μm mesh. Wash cells once with PBS buffer. Isolate CD8+ T cells using mouse CD8+ T cell enrichment isolation kit. Detailed steps are as follows: resuspend cells with 950 ul of 1 x MojoSort TM Buffer, add 50 ul antibody cocktail, mix and incubate on ice for 15 minutes. Add 50 ul magnetic beads, place on ice and mix every 5 minutes for a total of 15 minutes. Add 2 ml 1 x MojoSort TM Buffer, place on magnetic stand for 5 minutes and pour out the liquid containing CD8+ T cells. After centrifugation and counting, plate 3 x 10 6 cells in a 6-well plate pre-coated with mouse anti-CD3 (5 μg / mL), followed by addition of mouse anti-CD28 (2.5 μg / mL) for activation.

[0160] Step 2: Day 2, collect and count CD8 + T cells. Culture CD8 6 T cells at a density of 1-3 x 10 + cells / mL in medium containing 20 ng / mL mouse IL-2. T cells can be used for subsequent experiments starting on day 6 after activation.

[0161] Step 3: If T cells are to be infected, after activation of T cells, plate 0.6 x 10 6 OT-1 cells in a six-well plate and add CD11a knock-out retrovirus and mouse CD8 + T cell medium at a 1:1 ratio, and 8 μg / mL of Polybrene. Then, centrifuge the culture plate at 32°C, 1100 g for 2.5 hours.

[0162] Step 4: On day 3 of infection, replace the medium for OT-1 cells with fresh medium. On days 5-6 of infection, sort the desired OT-1 cells using a flow sorter for subsequent experiments.

[0163] Example 5, in vitro co-culture experiment of tumor cells and immune cells

[0164] Human system: Construct in vitro NY-ESO-1 expressing SW480 and A498 tumor cell lines. Obtain CD8 +T cells, and construct CTL cells targeting NY-ESO-1. The tumor cell line and NY-ESO-1 CTL cells are mixed in a certain ratio, co-cultured in the same culture dish, and the bispecific antibody is added in a concentration gradient. Experimental and control groups are set up: in the experimental group, the bispecific antibody CmScFv x AL-57 is additionally added to the co-cultured system; in the control group, the bispecific antibody CmScFv is additionally added to the co-cultured system. After co-culturing for a period of time, the expression of IFNg by T cells after co-culturing is counted by flow cytometry.

[0165] Mouse system: MC38 and B16-F10 tumor cell lines expressing EGFR are constructed in vitro and are referred to as MC38-EGFR and B16F10-EGFR, respectively. OT1 T cells are obtained by isolating the spleen of a mouse. The tumor cell line and OT1 T cells are mixed in a certain ratio, co-cultured in the same culture dish, and the bispecific antibody is added in a concentration gradient. Experimental and control groups are set up: in the experimental group, the OVA protein peptide (amino acid sequence: SIINFEKL, 100 ng / ml) is additionally added to the co-cultured system and the bispecific antibody Cet x ICAM1-D1 is added; in the control group, the OVA protein peptide (amino acid sequence: SIINFEKL, 100 ng / ml) is additionally added to the co-cultured system and the bispecific antibody Cetuximab is added. After co-culturing for a period of time, the expression of IFNg by T cells after co-culturing is counted by flow cytometry.

[0166] To evaluate the ability of the bispecific antibody to activate T cells, CD3, control antibody and bispecific antibody are coated separately or in combination in the cell plate, and then human CD8 + T cells are added, and the expression of T cell CD69 and IFN-γ is detected. Only when the bispecific antibody is combined with the anti-CD3 antibody can it further activate T cells, which shows that: 1) the deletion of the bispecific antibody is achieved by providing a costimulatory signal to activate T cells; 2) the bispecific antibody that provides a costimulatory signal is completely dependent on tumor antigen-specific activation and can to some extent avoid non-specific activation (corresponding to the shortcomings of the prior art).

[0167] Figures 4A and 4B show that the bispecific antibody in the present application can activate T cells through a costimulatory signal.

[0168] In the in vitro co-culture experiment, NY-ESO-1 tumor cells with ICAM-1 knock-out (sgICAM-1) and NY-ESO-1 specific CD8 +T cells were co-cultured in the presence of bispecific antibody or control antibody. Bispecific antibody significantly increased CD8 + T cell-mediated cytotoxicity, which was not observed with cetuximab or AL-57 antibody alone (Figure 5). This result demonstrates the feasibility and importance of anti-TAA and AL-57 Fab fragment in reconstituting ICAM-1 / LFA-1 signaling axis at the tumor-immune cell interface.

[0169] In addition, bispecific antibody significantly increased CD8 + IFN-γ expression in T cells, reaching a level similar to that observed in co-culture with wild-type SW480 and A498 tumor cells (Figure 6). This demonstrates that bispecific antibody can mimic tumor co-stimulatory molecule ICAM-1, bind to its ligand LFA-1, and compensate for the loss of ICAM-1 on the surface of tumor cells, avoiding tumor immune escape mediated by ICAM-1 loss.

[0170] NK cells are also regulated by a series of co-stimulatory molecules or co-inhibitory molecules on their surface. When the co-stimulatory molecule activation signal is stronger than the co-inhibitory molecule, NK cells can be activated and exert killing effect. In this application, NK cells isolated from PBMC were used to repeat the same experiment as in Figure 5, and similar results were found. Bispecific antibody significantly increased NK cell-mediated cytotoxicity, and reached a level similar to that observed in co-culture with ICAM-1-expressing normal tumor cells (Figure 7).

[0171] Figure 8 shows that the bispecific antibody in this application can enhance NK cell-mediated killing of ICAM-1 -deficient tumor cells. Different antibodies were added to the tumor and NK cell co-culture system at the same concentration. Then the proportion of viable cells after NK cell killing of tumor was detected by flow cytometry as an indicator of killing.

[0172] Example 6

[0173] This example provides another structure of bispecific antibody LFA-1 engager (Cet x ICAM1-D1), which is composed of Fab fragment of cetuximab and extracellular D1 domain of murine or human ICAM-1, and is fused to human Fc fragment containing "LALA-PG" mutation, and the structure and working schematic diagram are shown in Figure 9, wherein the Fab fragment is cetuximab targeting EGFR on the surface of tumor cells, and the Fc end is D1 domain of ICAM-1 targeting LFA-1 on the surface of T cells.

[0174] ICAM-1 D1 domain protein sequence:

[0175] "LALA-PG" human IgG Fc fragment protein sequence:

[0176] 1. Antibody affinity test of bispecific antibody

[0177] The tumor cells overexpressing EGFR were incubated with bispecific antibody, then stained with anti-human IgG antibody, and the mean fluorescence intensity was determined using flow cytometry. The half maximal effective concentration (EC50) of the bispecific antibody was 2.10 nM in this experiment (Figure 10).

[0178] 2. Activation of T cells by antibodies in vitro

[0179] In the in vitro co-culture experiment, B16F10 and MC38 tumor cells overexpressing EGFR were co-cultured with a gradient concentration of bispecific antibody or control antibody, respectively. Compared with the control antibody, the bispecific antibody significantly increased the expression of CD8 + IFN-γ in T cells, reaching a level similar to that observed in B16F10 and MC38 cells overexpressing ICAM-1 (Figure 11). This indicates that the bispecific antibody can compensate for the loss of ICAM-1 molecules on the surface of tumor cells, avoiding tumor immune escape mediated by ICAM-1 loss.

[0180] Example 7: Bispecific antibody treatment experiment in tumor-bearing mice

[0181] 1 x 10 6 MC38-EGFR and B16F10-EGFR cells were resuspended in PBS and subcutaneously inoculated into the right leg of mice. When the tumor was palpable, its length and width were measured with a vernier caliper. When the tumor volume was about 100 mm 3 The tumor-bearing mice were randomly divided into groups, ensuring that the tumor volumes in each group were similar. Subsequently, the mice were treated with Cetuximab, Cet x ICAM1-D1, and anti-PD-1 monoclonal antibody (Bio X Cell, Cat# BE0273, RRID: AB_2687796) on days D7, D10, and D13 (5 mg / kg of each antibody). Among them, when the bispecific antibody was treated, according to the experimental requirements, after the mice were anesthetized with isoflurane, the administration method and dose of each antibody were as follows: 5 mg / kg of Cet x ICAM1-D1 bispecific antibody was injected intratumorally or intravenously; anti-PD-1 treatment was 5 mg / kg of anti-PD-1 monoclonal antibody injected intraperitoneally. When the tumor volume reached 2000 mm 3 or the mice died, the experiment was terminated.

[0182] The mouse cell co-culture system experiment was performed according to the method of Example 5. The experimental results can be seen in Figure 12. In the in vitro co-culture experiment, only the co-culture system with the / SIINFEKL peptide treatment group detected the Cet x ICAM1-D1 bispecific antibody to promote the secretion of IFN-γ by OT-1 cells, which indicates that the LFA-1 engager Cet x ICAM1-D1 of the present application functions in a TCR-MHC signal-dependent manner. 257-264 The mouse cell co-culture system experiment was performed according to the method of Example 5. The experimental results can be seen in Figure 12. In the in vitro co-culture experiment, only the co-culture system with the / SIINFEKL peptide treatment group detected the Cet x ICAM1-D1 bispecific antibody to promote the secretion of IFN-γ by OT-1 cells, which indicates that the LFA-1 engager Cet x ICAM1-D1 of the present application functions in a TCR-MHC signal-dependent manner.

[0183] The mouse cell co-culture system experiment was performed according to the method of Example 5. The experimental results can be seen in Figure 13. In the in vitro co-culture experiment, the OT-1 cells with LFA-1 molecule deletion failed to express the increased expression of IFN-γ caused by the addition of the LFA-1 engager to the co-culture system. This indicates that the LFA-1 engager Cet x ICAM1-D1 functions in a LFA-1 molecule-dependent manner.

[0184] The human cell co-culture system experiment was performed according to the method of Example 5, and the A498 renal cancer cell line and SW480 colorectal adenocarcinoma cell line expressing endogenous EGFR were co-cultured with the NY-ESO-1 TCR T cells in Example 3. The experimental results can be seen in Figure 14. In the in vitro co-culture experiment, the human version of the LFA-1 engager Cet x hICAM1-D1 significantly enhanced the expression of IFN-γ in CD8 + T cells. This indicates that the human version of the LFA-1 engager can also remodel the ICAM-1 / LFA-1 signal axis in human tumors and T cells, and enhance the cytotoxicity of T cells.

[0185] The anti-tumor immune function of the LFA-1 engager Cet x ICAM1-D1 in vivo was verified in a mouse tumor-bearing model according to the method of Example 7. The local administration method of intratumoral injection was used. The experimental results can be seen in Figure 15. The tumors of MC38 and B16F10 after LFA-1 engager treatment were significantly inhibited in wild-type mice, and the tumor volume was significantly smaller than that of the Cetuximab treatment group, but there was no significant difference in the growth of the tumors in immune-deficient mice. This indicates that the LFA-1 engager functions to exert anti-tumor function through the body's immune system.

[0186] The anti-tumor immune function of the LFA-1 engager Cet x ICAM1-D1 in vivo was verified in a mouse tumor-bearing model according to the method of Example 7. The experimental results can be seen in Figure 16. When Cet x ICAM1-D1 was combined with anti-PD-1 for treatment, the tumor volume of the tumor-bearing mice was significantly smaller than that of the Cet x ICAM1-D1 treatment or anti-PD-1 treatment alone, and the tumor growth was significantly inhibited. This indicates that the LFA-1 engager can be combined with anti-PD-1 for treatment, and exhibits a better anti-tumor effect.

[0187] Example 8, Tumor infiltrating immune cell single cell transcriptome sequencing

[0188] MC38-EGFR tumor-bearing mice treated with Cetuximab and CetxICAM1-D1 bispecific antibody in Example 7 were euthanized, and tumors were dissected and prepared into single cell suspension. After staining with Zombie NIR and anti-mouse CD45, live CD45 + cells were sorted, and the same number of cells were collected for each group. After washing, single cell transcriptome library construction and high-throughput sequencing were performed.

[0189] Single Cell Experiment (V1.16.0) was used to perform quality control on single cell transcriptome sequencing data. After removing low-quality cells, the data was aligned to mouse reference genome (GRCm38 / mm10) and unique mapping reads were retained. Seurat (V5) was used to perform data integration, normalization, dimension reduction, clustering, and UMAP visualization, and marker genes were detected. The clustering results were manually annotated, and finally custom R (V4.1.3) scripts were used for data visualization.

[0190] Figure 17 shows single cell transcriptome analysis of T cells within MC38 tumors after treatment with LFA-1 engager CetxICAM1-D1 and Cetuximab of the present application.

[0191] Figure 18 shows changes in the level of T cell subsets within MC38 tumors after treatment with LFA-1 engager CetxICAM1-D1 and Cetuximab of the present application.

[0192] Figure 17 and Figure 18 show that LFA-1 engager promotes the conversion of tumor CD8 + T cells from precursor type to effector type T cells, indicating that LFA-1 engager can remodel CD8 + T cell subsets in the tumor microenvironment and stimulate their anti-tumor effector functions.

[0193] The above embodiments are only used to illustrate the present application, but not to limit the present application, and those skilled in the art can make various changes and modifications without departing from the essence and scope of the present application, for example, the structure of nano-antibody can be used, and similar antibodies designed based on the target of the present application. Other forms of multi-specific engager can also be designed based on RNA-delivery, for example, nanobody-based design, which contains the domain combination of TAA, AL-57, ICAM-1D1. Therefore, all equivalent technical solutions also belong to the protection scope of the present application.

Claims

1. A multispecific antibody or antigen-binding fragment thereof, comprising at least two segments of domains, wherein, One segment targets a relevant antigen or personalized surface antigen expressed on tumor cells, and the other segment targets LFA-1.

2. The multispecific antibody or antigen-binding fragment thereof of claim 1, wherein, The tumor-associated antigen includes, but is not limited to, one or more of an antigen of a relevant tumor expressing EGFR, an antigen of a relevant tumor expressing HER2, an antigen of a relevant tumor expressing PDL1, an antigen of a relevant tumor expressing CD19, an antigen of a relevant tumor expressing CD20, an antigen of a relevant tumor expressing CLL1, an antigen of a relevant tumor expressing CD22, an antigen of a relevant tumor expressing CD30, an antigen of a relevant tumor expressing BCMA, an antigen of a relevant tumor expressing EGFRvIII, an antigen of a relevant tumor expressing PSMA, an antigen of a relevant tumor expressing Muc1, an antigen of a relevant tumor expressing Claudin7, an antigen of a relevant tumor expressing TSA, an antigen of a relevant tumor expressing MSLN, an antigen of a relevant tumor expressing GPC3, an antigen of a relevant tumor expressing IL13RA2, an antigen of a relevant tumor expressing SLAMF7, an antigen of a relevant tumor expressing GPRC5D, an antigen of a relevant tumor expressing LILRB4, an antigen of a relevant tumor expressing DLL3, an antigen of a relevant tumor expressing TROP2, an antigen of a relevant tumor expressing Claudin 6, an antigen of a relevant tumor expressing B7-H3, an antigen of a relevant tumor expressing FAP, an antigen of a relevant tumor expressing CD123, and an antigen of a relevant tumor expressing Claudin18.

2.

3. The multispecific antibody or antigen-binding fragment thereof of claim 2, wherein: The relevant tumor expressing EGFR includes, but is not limited to, colorectal cancer, kidney cancer, non-small cell lung cancer, pancreatic cancer, nasopharyngeal cancer, head and neck tumor, etc. The relevant tumor expressing HER2 includes, but is not limited to, breast cancer, ovarian cancer, endometrial cancer, fallopian tube cancer, gastric cancer, and prostate cancer, etc. The relevant tumor expressing CD19 includes, but is not limited to, acute lymphoblastic leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, etc. The relevant tumor expressing CD20 includes, but is not limited to, acute lymphoblastic leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, etc. The relevant tumor expressing Claudin18.2 includes, but is not limited to, gastric cancer / gastroesophageal junction cancer, breast cancer, colon cancer, head and neck cancer, bronchial cancer, and non-small cell lung cancer, etc.

4. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein, The tumor-associated antigen is selected from one or more of CLL1, CD19, CD20, CD22, CD30, BCMA, EGFR, EGFRvIII, PSMA, Muc1, Claudin7, TSA, MSLN, GPC3, IL13RA2, SLAMF7, GPRC5D, LILRB4, DLL3, TROP2, PDL1, Claudin6, B7-H3, FAP, CD123, Claudin18.

2. Preferably, the tumor associated antigen is from an EGFR-expressing tumor of epithelial origin or a HER2-expressing tumor.

5. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein, The domain targeting a tumor associated antigen is from an amino acid sequence of an antibody or antigen binding fragment thereof that can bind the tumor associated antigen.

6. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein, The domain targeting a tumor associated antigen is from an EGFR antibody.

7. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein, The domain targeting a tumor associated antigen comprises a first heavy chain variable region VH1 comprising HCDR 1-3 having the amino acid sequences of SEQ ID NO. 1-3, respectively, and a first light chain variable region VL1 comprising LCDR 1-3 having the amino acid sequences of SEQ ID NO. 4-6, respectively: HCDR 1: NYGVH (SEQ ID NO. 1) HCDR 2: VIWSGGNTDYNTPFTS (SEQ ID NO. 2) HCDR 3: ALTYYDYEFAY (SEQ ID NO. 3) LCDR 1: RASQSIGTNIH (SEQ ID NO. 4) LCDR 2: YASESIS (SEQ ID NO. 5) LCDR 3: QQNNNWPTT (SEQ ID NO. 6).

8. The multispecific antibody or antigen binding fragment thereof of claim 7, wherein, the first heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% sequence identity to SEQ ID NO. 7; the first light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO.

8.

9. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein, The domain targeting a tumor associated antigen comprises a scFv fragment.

10. The multispecific antibody or antigen-binding fragment thereof of claim 9, wherein, The domain targeting a tumor associated antigen comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO.

9.

11. The multispecific antibody or antigen-binding fragment thereof of claim 7 or 9, wherein, The domain targeting a tumor associated antigen comprises an amino acid sequence of Cetuximab or an antigen binding fragment thereof.

12. The multispecific antibody or antigen-binding fragment thereof of any one of claims 1-11, wherein, The domain targeting LFA-1 has a sequence from an LFA-1 binding protein or from the D1 domain of ICAM-1.

13. The multispecific antibody or antigen-binding fragment thereof of claim 12, wherein, The LFA-1 binding protein is an LFA-1 agonistic protein.

14. The multispecific antibody or antigen-binding fragment thereof of claim 12 or 13, wherein, The LFA-1 binding protein is an antibody that can bind LFA-1.

15. The multispecific antibody or antigen-binding fragment thereof of claim 13 or 14, wherein, The LFA-1 binding protein is selected from one or more of the following proteins: AL57 antibodies such as CmScFvXAL-57, CBR LFA-1 / 2, CBR LFA-1 / 7, MEM83, TS2 / 4; 7E4, R2E7B, 17MEM48, MEM148, anti-phospho-b1 Thr-788 / 789, 12G10-488; TS1 / 22, TS1 / 18, anti-a4; MHM23; MHM24; CetXICAM1-D1.

16. The multispecific antibody or antigen-binding fragment thereof of claim 12, wherein, the second heavy chain variable region VH2 comprising the CDR sequences of a heavy chain as set forth in SEQ ID NO. 10; and the second light chain variable region VL2 comprising the CDR sequences of a light chain as set forth in SEQ ID NO.

11.

17. The multispecific antibody or antigen-binding fragment thereof of claim 16, wherein, the second heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% sequence identity with SEQ ID NO. 10; the second light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity with SEQ ID NO.

11.

18. The multispecific antibody or antigen-binding fragment thereof of claim 12, wherein, the second heavy chain variable region VH2 comprising the CDR sequences of a heavy chain as set forth in SEQ ID NO. 10; and the second light chain variable region VL2 comprising the CDR sequences of a light chain as set forth in SEQ ID NO.

11. Preferably, the multispecific antibody or antigen-binding fragment thereof comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.4%, at least 99.6%, at least 99.7%, at least 99.8%, or 100% sequence identity with SEQ ID NO.

13.

19. The multispecific antibody or antigen-binding fragment thereof of claim 1, wherein, The personalized surface antigen is the epithelial cell marker EpCAM.

20. The multispecific antibody or antigen-binding fragment thereof of any one of claims 1-19, which is a bispecific antibody.

21. The multispecific antibody or antigen-binding fragment thereof of claim 20, which is a bispecific antibody targeting LFA-1 and EGFR.

22. A nucleic acid molecule encoding the multispecific antibody or antigen-binding fragment thereof of any one of claims 1-21.

23. An expression vector comprising the nucleic acid molecule of claim 22.

24. A recombinant cell comprising the nucleic acid molecule of claim 22.

25. A method of producing the multispecific antibody or antigen-binding fragment thereof of any one of claims 1-21, the method comprising: constructing a plasmid comprising a nucleic acid sequence that encodes the first domain and a plasmid comprising a nucleic acid sequence that encodes the second domain; Alternatively, a plasmid comprising a nucleic acid sequence encoding the first domain and a nucleic acid sequence encoding the second domain is constructed; transfecting the plasmid into a host cell to produce a fusion protein comprising the first domain and the second domain, thereby obtaining the multispecific antibody.

26. Use of the multispecific antibody or antigen-binding fragment thereof of any one of claims 1-21 in the manufacture of a medicament for treating a tumor.

27. The use according to claim 26, wherein, The tumor includes one or more of the following: an EGFR-expressing related tumor, a HER2-expressing related tumor, a PD-L1-expressing related tumor, a CD19-expressing related tumor, a CD20-expressing related tumor, a CLL1-expressing related tumor, a CD22-expressing related tumor, a CD30-expressing related tumor, a BCMA-expressing related tumor, a EGFRvIII-expressing related tumor, a PSMA-expressing related tumor, a Muc1-expressing related tumor, a Claudin7-expressing related tumor, a TSA-expressing related tumor, a MSLN-expressing related tumor, a GPC3-expressing related tumor, a IL13RA2-expressing related tumor, a SLAMF7-expressing related tumor, a GPRC5D-expressing related tumor, a LILRB4-expressing related tumor, a DLL3-expressing related tumor, a TROP2-expressing related tumor, a Claudin 6-expressing related tumor, a B7-H3-expressing related tumor, a FAP-expressing related tumor, a CD123-expressing related tumor, and a Claudin18.2-expressing related tumor.

28. The use of claim 27, wherein: The EGFR-expressing related tumor includes but is not limited to: colorectal cancer, kidney cancer, non-small cell lung cancer, pancreatic cancer, nasopharyngeal cancer, head and neck tumor, etc. The HER2-expressing related tumor includes but is not limited to: breast cancer, ovarian cancer, endometrial cancer, fallopian tube cancer, gastric cancer, and prostate cancer, etc. The CD19- and CD20-expressing related tumor includes but is not limited to: acute lymphoblastic leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, etc. The Claudin18.2-expressing related tumor includes but is not limited to: gastric cancer / gastroesophageal junction cancer, breast cancer, colon cancer, head and neck cancer, bronchial cancer, and non-small cell lung cancer, etc.

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