CN115925942A - Nano antibody targeting human LILRB4 and application thereof - Google Patents

Abstract

The invention discloses a nano antibody targeting LILRB4, a nucleic acid encoding the nano antibody, an expression vector containing the nucleic acid, a pharmaceutical composition containing the nano antibody and an application of preparing a medicament.

Description

Nano antibody targeting human LILRB4 and application thereof

Technical Field

The invention belongs to the field of antibody engineering, particularly relates to a therapeutic single-domain antibody for diagnosing or treating tumors, and particularly relates to a nano antibody targeting LILRB4, a derivative protein thereof and application thereof in preparing a medicament.

Background

Nanobodies are the smallest antibody molecule at present, originally found in camelid blood by the belgium scientist Hamers, a class of great interest in engineered antibody products. The nano antibody has the main advantages that: firstly, the volume is 1/10 of that of the common antibody, because of the small volume, the penetrating power of the antibody in animal tissues is strong, for example, the antibody can reach the interior of a high-density tumor through the brain tissues of a human body, but the common antibody cannot, so that certain tumors or brain diseases can be treated through the nano antibody; secondly, the antigen specificity is good; thirdly, the gene modification is easy, and the artificial modification is convenient to obtain antibodies for resisting different pathogens; fourthly, the stability is high, for example, the nano antibody is not naturally decomposed in vivo for a longer time than the common antibody (meaning that the drug effect time is longer), and the nano antibody can even pass through the human stomach to keep effectiveness.

LILRB4 (also known as ILT3, LIR5, CD 85K) is a member of the immunoglobulin-like receptors of leukocytes (LILRs/LIRs), an immunomodulatory transmembrane protein found on Antigen Presenting Cells (APC). LILRB4 inhibits APC activation and induces immune tolerance by T suppressor cells. It has been shown to modulate the immune response by inducing T cell anergy and differentiation of CD8+ T suppressor cells, and may play a role in establishing immune tolerance against cancer. It is expressed mainly in tolerant Dendritic Cells (DCs), myeloid-derived suppressor cells and M2 macrophages, with a small amount expressed on the surface of plasma cells and not on the surface of hematopoietic precursor cells or stem cells. LILRB4 is a marker molecule for monocytic acute myeloid leukemia (monocytic AML) and is highly expressed on the surface of the monocytic AML cells.

Currently, 2 antibody drugs targeting LILRB4 are in clinical research stage, NGM831 developed by NGM Biopharmaceuticals is currently in the first clinical stage, and the indications are pancreatic cancer, breast cancer, gastric cancer, non-small cell lung cancer, cervical cancer, endocervical cancer, squamous cell carcinoma of head and neck, bladder urothelial cancer, colorectal cancer, esophageal cancer, ovarian cancer, renal cell carcinoma, prostate cancer, melanoma, mesothelioma, bile duct cancer and the like; IO-202 developed by Immune-on Therapeutics is in the first clinical stage at present, and indications comprise acute myelogenous leukemia, myelomonocytic leukemia and the like.

Disclosure of Invention

In the prior art, scFv, fab or whole IgG anti-LILRB 4 antibody molecules have complex structures and larger molecules, and although active molecules can be connected to LILRB4, the functional and method influencing the active molecules are complex and the load efficiency is lower; the nano antibody has small molecules and is easy to operate, but the humanized degree is low, the affinity is not high, and the half-life prolonging property needs to be further improved.

Aiming at the defects of the prior art, the invention provides a series of antihuman LILRB4 nano antibody sequences and a preparation scheme thereof. The provided LILRB 4-resistant nano antibody can be combined with human LILRB4 with high affinity, and can definitely observe the activation effect on T cells on a cytological model, thereby having potential therapeutic value on tumors.

In a first aspect, the present invention provides a nanobody against LILRB4, which is capable of specifically binding to LILRB4 according to an embodiment of the present invention, and in which complementarity determining region CDRs of VHH chains are one or more selected from the group consisting of:

(1) SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, CDR3;

(2) SEQ ID NO:19, CDR1 shown in SEQ ID NO:20, and SEQ ID NO:21 CDR3;

(3) SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, CDR3;

(4) SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, CDR3;

(5) The amino acid sequence of SEQ ID NO:28, CDR1 shown in SEQ ID NO:29, and the CDR2 shown in SEQ ID NO: CDR3 shown in FIG. 30;

(6) The amino acid sequence of SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:31, CDR3;

(7) The amino acid sequence of SEQ ID NO:32, CDR1 shown in SEQ ID NO:33, and the CDR2 shown in SEQ ID NO:34 CDR3;

(8) The amino acid sequence of SEQ ID NO:35, SEQ ID NO:36, and the CDR2 shown in SEQ ID NO:37, CDR3 shown.

Further, in some embodiments of the invention, the nanobody is a humanized VHH or a camelized VH.

Further, in some embodiments of the present invention, the nanobody has an amino acid sequence as set forth in SEQ ID NO: 3. 4, 5, 6, 7, 8, 9, 10, 11, 12, 14 and 15.

In a second aspect, the present invention provides a fusion protein, according to an embodiment of the present invention, comprising a functional domain capable of specifically binding to LILRB4, the functional domain consisting of any one of the nanobodies against LILRB4 described above.

The nano antibody provided by the invention can be fused with any other protein or substance to achieve different purposes, such as combining with fluorescent protein, enzyme or radioactive element to achieve the purpose of easy detection, and further fusing with drug molecules for treating LILRB4 mediated related diseases to achieve better treatment purpose. The type of protein fused with the nanobody can be reasonably selected by those skilled in the art according to actual needs or purposes, and it is also within the scope of the present invention to fuse whatever type of substance is fused.

In a third aspect, the present invention provides an anti-LILRB 4 antibody, according to an embodiment of the present invention, the antibody is a conventional antibody or a functional fragment thereof, and the heavy chain variable region of the antibody is composed of any one of the aforementioned nanobodies against LILRB 4;

further, the functional fragment is the Fab, fab ', (Fab') 2, fv, scFv or sdFv structure of the conventional antibody.

Traditional antibodies consist structurally of two identical heavy chains and two identical light chains, a light chain having a light chain variable region (VL) and a light chain constant region (CL); the heavy chain has a heavy chain variable region (VH) and heavy chain constant regions (CH 1, CH2, CH3 and/or CH 4). On the premise that the present invention discloses a structure of a nanobody capable of specifically binding to LILRB4, those skilled in the art can easily think of using the nanobody of the present invention to modify a conventional antibody, for example, applying the CDR region structure of the nanobody of the present invention to the conventional antibody to obtain the conventional antibody capable of specifically binding to LILRB4, and such conventional antibodies also belong to the protection scope of the present invention; further, based on the structure of the conventional antibody, a partial structure thereof such as a Fab, fab ', (Fab') 2, fv, scFv or sdFv structure, etc. also has the binding specificity of LILRB4, which also falls within the scope of the present invention.

In a fourth aspect, the present invention provides a composition for treating diseases, which comprises the nanobody against LILRB4 described in any one of the above, the fusion protein described above or the antibody described above, and a pharmaceutically acceptable excipient.

The pharmaceutically acceptable auxiliary materials refer to those in the pharmaceutical field, such as: diluents, fillers, binders, wetting agents, absorption enhancers, surfactants, disintegrants, adsorption carriers, lubricants, and the like. In addition, other adjuvants such as flavoring agent and sweetener can also be added. The auxiliary materials are one or more than two of diluent, filler, adhesive, wetting agent, absorption enhancer, surfactant, disintegrant, adsorption carrier, lubricant, flavoring agent and sweetener.

The dosage form of the medicament provided by the invention is not strictly limited, and the medicament can be prepared into various dosage forms according to the existing method in the field of medicaments, and is applied to patients needing treatment in the modes of oral administration, nasal inhalation, rectum, parenteral administration or transdermal administration and the like.

In a fifth aspect, the invention provides an isolated nucleic acid molecule encoding a single domain antibody as described in any one of the above.

Based on the disclosure of the present invention, the polynucleotide molecules encoding the nanobodies and the fusion proteins are easily obtained by those skilled in the art through the conventional techniques in the art, and based on the degeneracy of the codon, the polynucleotide molecules are varied, and there are many possibilities for the specific base sequences thereof, and therefore, it is within the scope of the present invention to provide the polynucleotide molecules with the capability of encoding the single domain antibodies or fusion proteins of the present invention regardless of the variation of the polynucleotide molecules.

In a sixth aspect, the present invention provides a vector comprising a nucleic acid molecule as described above.

In a seventh aspect, the present invention provides a recombinant cell comprising a vector as described above.

In an eighth aspect, the present invention provides a method of preparing a nanobody as described in any one of the above, comprising: culturing the recombinant cells, and separating and purifying the culture product to obtain the nano antibody.

It should be noted that the preparation of the nanobody, fusion protein or antibody of the present invention may be achieved by chemical synthesis, genetic engineering techniques, or other methods, and all of the methods for preparing the nanobody, fusion protein or antibody of the present invention are within the scope of the present invention.

In a ninth aspect, the present invention provides a method of activating a T cell, comprising contacting a T cell with an anti-LILRB 4 nanobody as described in any of the above, a fusion protein as described above, an antibody as described above, a composition as described above, a nucleic acid molecule as described above, a vector as described above, or a recombinant cell as described above.

Optionally, the contacting comprises in the presence of a cancer cell.

LILRB4 is expressed on monocytes, macrophages and dendritic cells, and can suppress innate immunity in a cell-autonomous manner and inhibit T cell activation through an indirect mechanism. LILRB4 is a specific marker for monocytic Acute Myeloid Leukemia (AML), including refractory and relapsed diseases. LILRB4 has been shown to support tumor cell infiltration into tissues and inhibit T cell activity through signaling pathways involving APOE, LILRB4, SHP-2, uPAR and ARG1 in AML cells (Deng m.et ah, nature (2018) 562.

In a tenth aspect, there is provided a use of the anti-LILRB 4 nanobody, the fusion protein as described above, the antibody as described above, the composition as described above, the nucleic acid molecule as described above, the vector as described above, or the recombinant cell as described above of the present invention, for preparing a medicament for treating a related disease mediated by human LILRB4 protein.

The antibody of the invention can be used for treating the LILRB4 target neutralization LILRB4 to realize the effect of preventing, treating and/or improving diseases, such as but not limited to tumors.

To better understand the present invention, certain terms are first defined. Other definitions are listed throughout the detailed description section.

In general, the antigen binding properties of an antibody can be described by 3 specific regions, called variable regions (CDRs), located in the heavy chain variable region, which are separated into 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction. These CDRs form a loop structure, and the β -sheets formed by the FRs between them are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of antibodies of the same type.

The invention includes not only intact antibodies, but also fragments of antibodies with immunological activity or fusion proteins of antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.

"chimeric antibody" refers to an antibody in which the amino acid sequences of immunoglobulin molecules are derived from two or more species. Typically, the variable regions of both the light and heavy chains correspond to those of an antibody derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capacity, while the constant regions are homologous to sequences in the antibody derived from another species (typically human) to avoid eliciting an immune response in that species.

"nanobodies" are generally as defined in WO 2008/020079 or WO 2009/138519, and thus in one particular aspect generally represent VHHs, humanized VHHs or camelized VH (such as camelized human VH), or generally sequence optimized VHHs (e.g. optimized for chemical stability and/or solubility, maximal overlap with known human framework regions and maximal expression). The 'nano antibody' is obtained by a genetic engineering method, and mainly has 3 types, wherein the first type is a heavy chain variable region obtained from a camelid HCAb, is a single folding unit, retains complete antigen binding activity, and is a minimum natural antibody fragment. The second type is a heavy chain variable region obtained from IgNAR of cartilaginous fish such as shark, which is denoted by VNAR. The third type is a heavy chain or light chain variable region obtained from a monoclonal antibody of human or murine origin, which retains antigen binding activity but has greatly reduced affinity and solubility.

"Fc region" or "Fc" refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the hinge region, a CH2 domain, and a CH3 domain, which mediates binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component of the classical complement system (e.g., C1 q), including native sequence Fc regions and variant Fc regions. Typically, the human IgG heavy chain Fc region is the carboxy-terminal stretch from the amino acid residue at position Cys226 or Pro230, but the boundaries may vary. The C-terminal lysine of the Fc region (residue 447, according to the EU numbering system) may or may not be present. Fc may also refer to this region of sequestration, or in the case of Fc-containing protein polypeptides, such as "binding proteins comprising an Fc region," also referred to as "Fc fusion proteins" (e.g., antibodies or immunoadhesins). The native sequence Fc region in the antibodies of the invention includes human IgG1, igG2 (IgG 2A, igG 2B), igG3 and IgG4. In IgG, igA, and IgD antibody isotypes, the Fc region comprises the CH2 and CH3 constant domains of each of the two heavy chains of an antibody; the IgM and IgEFc regions comprise three heavy chain constant domains (CH domains 2-4) in each polypeptide chain.

By "specific binding" is meant a non-random binding reaction between two molecules, such as between an antibody and the antigen to which it is directed. The term "immunological binding" refers to a specific binding reaction that occurs between an antibody molecule and an antigen for which the antibody is specific. The strength or affinity of an immunological binding interaction may be expressed in terms of the equilibrium dissociation constant (KD) of the interaction, where a smaller KD value indicates a higher affinity. The immunological binding properties between the two molecules can be quantified using methods well known in the art. One method involves measuring the rate of antigen binding site/antigen complex formation and dissociation. Both the "association rate constant" (Ka or Kon) and the "dissociation rate constant" (Kd or Koff) referring to a particular antibody-antigen interaction can be calculated by concentration and the actual rate of association and dissociation, and Kd, ka and Kd values can be measured by any effective method. In a preferred embodiment, the dissociation constant is measured by bioluminescence interferometry. In other preferred embodiments, the dissociation constant can be measured using surface plasmon resonance techniques (e.g., biacore) or KinExa.

"vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and an episomal mammalian vector). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operatively linked.

"nucleic acid molecule" is intended to include both DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, and may be a cDNA.

The invention achieves the following beneficial technical effects:

the anti-LILRB 4 nano antibody can be combined with human LILRB4 with high affinity, and the activation effect on T cells can be clearly observed on a cytological model, so that the antibody has potential therapeutic value on tumors.

Drawings

Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. Also, like reference numerals are used to refer to like parts throughout the drawings. In the drawings:

FIG. 1: FACS detection of binding Activity of anti-human LILRB4 chimeric antibody on THP-1 cells

FIG. 2: FACS detection of binding Activity of anti-human LILRB4 chimeric antibody on RPMI-8226 cells

FIG. 3: activation profile of anti-human LILRB4 chimeric antibody on T cells

FIG. 4: FACS detection of binding Activity of anti-human LILRB4 humanized antibody on RPMI-8226 cells

Detailed Description

Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

Example 1: construction of camel nano antibody immune phage library

Utilizing antigen immune camel, separating Peripheral Blood Mononuclear Cells (PBMC), extracting total RNA for reverse transcription, amplifying a variable region (VHH) of a nanometer antibody heavy chain by using a reverse transcription product as a template, connecting the VHH with a phage display carrier, and electrically transferring Escherichia coli TG1 competent cells to construct a camel immune library.

Specifically, camels were immunized twice weekly for 4 times. Each injection of 0.8mg of LILRB4 extracellular domain recombinant protein, freund's complete/incomplete adjuvant (Sigma, F5881, F5506), adopt subcutaneous multiple injection. 1mL of blood serum was collected 2 weeks after each immunization, and total antibody (IgG) and heavy chain antibody (heavy chain antibody) in the serum were measured by ELISA using immunogen as the antigeny, hcAb). When the serum titer meets the requirement of library construction, collecting 100mL camel peripheral blood, separating PBMC by using a separation kit (Tianjin tertiary ocean, cat: TBD2011 CM), extracting the total RNA of the PBMC, inverting to obtain cDNA, and using the cDNA as a template for subsequent VHH fragment amplification. Searching camel source VHH antibody genes according to related literatures and databases, designing and synthesizing VHH antibody library construction primers, and amplifying antibody variable region gene sequences by PCR. The vector and amplified antibody fragment are then cleaved enzymatically using an endonuclease. The ligation product is constructed by adopting a T4 ligase ligation mode, and is transferred into TG1 strain by an electrotransfection technology. Finally construct a 1.8X 10 ⁸ A camel anti-human LILRB4 VHH antibody immune library is used for screening specific anti-human LILRB4 nano antibodies. In order to test the accuracy of the library, 50 clones were randomly selected for colony PCR, and the results showed that the insertion rate reached 90%.

And screening the constructed camel immune library by a solid-phase screening method to obtain the specific phage display nano antibody. 10 phage-displayed nanobodies which can simultaneously bind to the recombinant protein of human LILRB4 were obtained by original library presentation and screening and identification: d2, D5, C6, C7, H9, F1, C3, B2, E5 and B4.

Example 2: preparation of anti-human LILRB4 nano antibody and control antibody

Variable region gene synthesis is carried out on the same target point contrast antibody 193 (sequence source: WO2020056077A1, SEQ NO.231 and SEQ NO. 237), and the light chain variable region sequence and the heavy chain variable region sequence are shown as SEQ ID NO.1 and SEQ ID NO. 2. Respectively cloning the light and heavy chain sequences into eukaryotic transient expression vectors containing human kappa/IgG1 light and heavy chain constant regions to obtain control antibody light chain and heavy chain expression plasmids, transferring the control antibody light chain and heavy chain expression plasmids into escherichia coli for amplification, separating to obtain a large amount of plasmids containing the control antibody light chain and heavy chain, extracting the plasmids, performing ethanol precipitation, and respectively transferring the light and heavy chain plasmids of the control antibody into HEK293 cells for recombinant expression according to the operation instructions of a transfection reagent 293fectin the fields of Cat:12347019 and Gibco. 5-6 days after cell transfection, culture supernatant is taken and purified by a ProA affinity chromatography column to obtain a control antibody.

According to the sequencing result of the phage-displayed nanobody, primers are designed, D2, D5, C6, C7, H9, F1, C3, B2, E5 and B4 are cloned into a eukaryotic transient expression vector containing a human Fc (hFc) encoding gene through a PCR method, and are expressed in HEK293 cells in a recombination mode. After 5-6 days of cell transfection, culture supernatant is taken, and expression supernatant is purified by utilizing a ProA affinity chromatography column to obtain chD2, chD5, chC6, chC7, chH9, chF1, chC3, chB2, chE5 and chB4 recombinant proteins. The sequence of the chD2 variable region is shown in SEQ ID NO.3, the sequence of the chD5 variable region is shown in SEQ ID NO.4, the sequence of the chC6 variable region is shown in SEQ ID NO.5, the sequence of the chC7 variable region is shown in SEQ ID NO.6, the sequence of the chH9 variable region is shown in SEQ ID NO.7, the sequence of the chF1 variable region is shown in SEQ ID NO.8, the sequence of the chC3 variable region is shown in SEQ ID NO.9, the sequence of the chB2 variable region is shown in SEQ ID NO.10, the sequence of the chE5 variable region is shown in SEQ ID NO.11, the sequence of the chB4 variable region is shown in SEQ ID NO.12, and the sequence of the constant region is shown in SEQ ID NO.13.

SEQ ID NO.1:193 heavy chain variable region amino acid sequence

EVQLVESGGGLVQPGGSLRLSCAASGFSLSSSYWISWVRQAPGKGLEWIGSIDSGSVGITYYATWVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGDNWALDLWGQGTLVTVSS

SEQ ID NO.2:193 light chain variable region amino acid sequence

DIQMTQSPSTLSASVGDRVTITCRASQSINSWLAWYQQKPGKAPKLLIYKASTLASGVPSRFSGSGSGTEFTLTISSLQPDDAATYYCQHGYIRGDLDNVFGGGTKVEIK

Amino acid sequence of SEQ ID NO.3 chD2 VHH

QVQLQESGGGSVQAGGSLRLSCTASGYTASSDYMGWFRQAPGKKREGVACINTNGGETYHANSVSGRFTISRDNAKNTVYLQMNSLKPDDTAMYYCAVGRTNPDSYGGSRCLLAPEYTYWGQGTQVTVSS

The amino acid sequences of the chD2 antigen complementarity determining regions CDRs 1, 2 and 3 are respectively SEQ ID NO: 16. 17 and 18, underlined (according to the definition of Kabat CDRs).

Amino acid sequence of SEQ ID NO.4 chD5 VHH

QVQLQESGGGSVQAGGSLRLSCATSGDTYSTLCMGWFRQAPGKEREGVAAIYRGGDSTVYADSVKGRFTISQDNAKNTVYLQMNGLKPEDTAIYYCAASSLGRCAADIRTGPPFWAVGFRYWGQGTQVTVSS

The amino acid sequences of the chD5 antigen complementarity determining regions CDRs 1, 2 and 3 are respectively SEQ ID NO: 19. 20 and 21, underlined (according to the definition of the Kabat CDRs).

Amino acid sequence of SEQ ID NO.5 chC6 VHH

QVQLQESGGGLVQPGGSLRLSCAASGLAFSRYYMSWVRQAPGKGLEWVSGIRSDGLSTSYADSVKGRFTISRDNAKNTLFLQMNNLKSEDTALYYCATGVGDSGDYRGQGTQVTVSS

The amino acid sequences of the chC6 antigen complementarity determining regions CDRs 1, 2 and 3 are respectively SEQ ID NO: 22. 23 and 24, underlined (according to the definition of the Kabat CDRs).

chC7 VHH amino acid sequence of SEQ ID No.6

QVQLQESGGGSVQAGGSLRLSCAASEYTYSRHCMAWFRQAPGKEREGVATIYTGGGITRYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADIASRACVTDPFPLKHAQFSSWGQGTQVTVSS

The amino acid sequences of the chC7 antigen complementarity determining regions CDRs 1, 2 and 3 are respectively SEQ ID NO: 25. 26 and 27, underlined (according to the definition of the Kabat CDRs).

Amino acid sequence of SEQ ID NO.7 chH9 VHH

QVQLQESGGGSVQAGGSLRLSCAVSEYVYSRCTMAWYRQAPGKERELVSAFDSGETTWYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCNTVGVKSVKSGGGSWCFSPDYWGQGTQVTVSS

The amino acid sequences of the CDRs 1, 2 and 3 of the chH9 antigen complementarity determining region are SEQ ID NOs: 28. 29 and 30, underlined (according to the definition of Kabat CDRs).

Amino acid sequence of chF1 VHH of SEQ ID No.8

QVQLQESGGGLVQAGGSLRLSCAASEYSYRRHCMAWFRQAPGKEREGVATIYTGGGITRYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADIASRACVTDPFPLERARFSAWGQGTQVTVSS

The amino acid sequences of the chF1 antigen complementarity determining regions CDRs 1, 2 and 3 are respectively SEQ ID NO: 25. 26 and 31, underlined (according to the definition of the Kabat CDRs).

Amino acid sequence of SEQ ID NO.9 chC3 VHH

QVQLQESGGGLVQAGGSLRLSCVASEYIYTRCNMAWYRQAPGKERELVSAFDIGDTLYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCNTVGVKPGGGSWCFNPDYWGQGTQVTVSS

The amino acid sequences of the chC3 antigen complementarity determining regions CDRs 1, 2 and 3 are respectively SEQ ID NO: 32. 33 and 34, underlined (according to the definition of the Kabat CDRs).

SEQ ID NO.10 chB2 VHH amino acid sequence

QVQLQESGGGPVQAGGSLRLSCAASEYSYRRHCMAWFRQAPGKEREGVATIYTGGGITRYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADIASRACVTDPFPLERARFSAWGQGTQVTVSS

The amino acid sequences of the CDRs 1, 2 and 3 of the chB2 antigen complementarity determining region are SEQ ID NOs: 25. 26 and 31, underlined (according to the definition of the Kabat CDRs).

Amino acid sequence of chE5 VHH of SEQ ID NO.11

QVQLQESGGGLVQAGGSLRLSCAASEYTYSRHCMAWFRQAPGKEREGVATIYTGGGITRYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADIASRACVTDPFPLKHAQFSSWGQGTQVTVSS

The amino acid sequences of the chs 5 antigen complementarity determining regions CDRs 1, 2 and 3 are respectively SEQ ID NO: 25. 26 and 27, underlined (according to the definition of the Kabat CDRs).

Amino acid sequence of SEQ ID NO.12: chB4 VHH

QVQLQESGGGPVQAGGSLRLSCAASRYDIETKCITWLRQAPGKERERVASISPGDGSTYYADSVKGRFTISQEYAKNTVDLQMNSLKSEDTAMYYCAAARAPWGRCAQWTAGIDFDYWGQGTQVTVSS

The amino acid sequences of the CDRs 1, 2 and 3 of the chB4 antigen complementarity determining regions are SEQ ID NOs: 35. 36 and 37, underlined (according to the definition of the Kabat CDRs).

hFc constant region amino acid sequence of SEQ ID NO.13

ASEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Example 3: chimeric antibody affinity detection

The antibody affinity was determined by capturing the Fc fragment of the antibody with an Ocet QKe system instrument from Fortebio using an anti-human antibody Fc fragment capture Antibody (AHC) biological probe. For the measurement, the chD2, chD5, chC6, chC7, chH9, chF1, chC3, chB2, chE5 and chB4 chimeric antibodies and the control antibody 193 were diluted to 4ug/ml in PBS buffer and passed over the surface of an AHC probe (Cat: 18-0015, PALL) for 120 seconds. Human LILRB4 recombinant protein (purchased from Yiqiao, cat.16742-H08H) or cynomolgus monkey LILRB4 recombinant protein (purchased from ACRO, cat.3CDK-C5227) 60nm; the mobile phase had an association time of 300s and a dissociation time of 300s. After the experiment, blank control response values were deducted, and the software was run for 1:1Langmuir binding pattern was fitted and kinetic constants for antigen-antibody binding were calculated.

The kinetic parameters are shown in table 1 below, and the results show that the 10 chimeric antibodies all bind to human LILRB4 recombinant protein, wherein the affinity of chD2, chD5, chC6 is superior or equivalent to that of the control antibody 193. The 10 chimeric antibodies were all bound to cynomolgus monkey LILRB4 recombinant protein, and the affinity of the remaining 9 chimeric antibodies to cynomolgus monkey LILRB4 recombinant protein was superior or equivalent to that of the control antibody 193 except chC 6.

TABLE 1 affinity assay of chimeric antibodies to LILRB4 recombinant proteins

Example 4: FACS detection of binding Activity of anti-human LILRB4 chimeric antibody on THP-1 cells

2E5 THP-1 cells were combined with different concentrations of anti-LILRB 4 chimeric antibody, chD2, chD5, chC6, chC7, chH9 chimeric antibody and control antibody 193, and diluted with 3-fold gradient starting from 198nM for 11 gradients. Incubated at 4 ℃ in the dark for 60min, after sufficient PBS washing, FITC-labeled goat anti-human antibody (sigma, F9512) diluted at 1:200 was added, incubated at 4 ℃ in the dark for 30min, then sufficiently PBS washing was performed, resuspended in 200ul of PBS, and detected by flow cytometry.

The results show (FIG. 1), that chD2, chD5, chC6, chC7 and chH9 can be bound to THP-1 cells of human LILRB4 positive cells in a dose-dependent manner, the EC50 values are shown in Table 2, and the binding capacity on the THP-1 cells is better than that of a control antibody.

TABLE 2 FACS detection of the bound EC50 values of anti-human LILRB4 chimeric antibodies on THP-1 cells

	193	chD2	chD5	chC6	chC7	chH9
							EC50(nM)	1.43	0.27	0.31	0.22	0.39	0.44

Example 5: FACS detection of binding Activity of anti-LILRB 4 chimeric antibodies on RPMI-8226 cells

2E5 RPMI-8226 cells were combined with different concentrations of anti-LILRB 4 chimeric antibody, chF1, chC3, chB2, chE5, and chB4 chimeric antibody and control antibody 193, respectively, and diluted with 8 gradients starting from 132nM in 3-fold gradients. Incubated at 4 ℃ in the dark for 60min, washed thoroughly with PBS, then added with FITC-labeled goat anti-human antibody (sigma, F9512) diluted at 1.

The results show (fig. 2) that chF1, chC3, chB2, chE5 and chB4 can bind human LILRB4 positive cells RPMI-8226 cells dose-dependently with no weaker binding activity than control 193.

Example 6: LILRB4 cell model evaluation of T cell activation by anti-human LILRB4 chimeric antibody

Taking logarithmically grown APOE/TCR Activator/CHO cells, centrifuging for 5 minutes at 1000 rpm after trypsinization to remove supernatant, resuspending in fresh F12K medium containing 10% FBS, adjusting the density of resuspended cells to 4X 10 ⁵ And/ml. The resuspended cells were seeded into a 96-well white-walled clear-bottomed cell culture plate, 100 ul/well cell suspension, 5% CO2 incubator at 37 ℃ overnight. The following day the APOE/TCR Activator/CHO cell-inoculated 96-well plates were blotted with F12K +10% FBS medium and the plates were rinsed once with 150 ul/well DPBS, blotted of DPBS, followed by gradient dilution of the chD2, chD5, chH9 chimeric antibody and the control antibody 193 with 0.5% BSA-containing RPMI1640 medium (starting at 256nM, 4-fold gradient dilution in sequence of 11 concentrations), addition of the gradient diluted 2 × concentration samples (50 ul/well) to the cell-inoculated 96-well plates, and additional antibody-free medium control wells. Taking log-phase grown LILRB4 Effect Reporter cells, centrifuging the cells to discard the supernatant, washing the cells once with DPBS, centrifuging to remove DPBS, then resuspending the cells in fresh RPMI1640 medium containing 0.5% BSA to adjust the density of the resuspended cells to 8X 10% ⁵ The cells were then added to the above antibody-containing 96-well plates at 50ul per well and placed in a 37 ℃ incubator for further incubation for 5 to 6 hours. The 96-well plate was removed from the incubator and 100 ul/well Bright-Glo was added ^TM The luciferase detection reagent is placed for 3 minutes and put into an enzyme-linked immunosorbent assay to read the value. From the corresponding readings for each gradient concentration well, a gradient curve of sample versus cell activation was fitted using Prism Graphpad software and the half effective binding concentration (EC 50) of the sample was calculated.

The activation gradient curves are shown in fig. 3, the corresponding EC50 s are shown in table 3, and the activating effect of chD2, chD5, chH9 on T cells is similar to that of control antibody 193.

TABLE 3 EC50 values for T cell activation by anti-human LILRB4 chimeric antibodies

	chD2	chD5	chH9		193
						EC50(nM)	0.153	0.043	0.064	0.094

Example 7: humanization and recombinant expression analysis of anti-human LILRB4 nano antibody

Firstly, the sequence of the camel source antibody VHH is comprehensively analyzed, and the antigen Complementary Determining Region (CDR) of the antibody combined with the antigen and a framework region (framework) supporting the conserved three-dimensional conformation of the antibody are determined. And then selecting the most similar humanized antibody template as a basic template according to the homology comparison result, and carrying out CDR grafting by combining the result of full sequence blast to realize the humanization of the chD2 variable region (VH) in the Framework region. The amino acid sequence of the D2 humanized molecule hzD2-1 is shown in a sequence 14; the D2 humanized molecule hzD2-2 amino acid sequence is shown in sequence 15. The humanized hzD2-1 and hzD2-2 variable region sequences are synthesized completely and cloned to eukaryotic expression vector containing human Fc (hFc) encoding gene. And after obtaining an expression plasmid with a correct sequence, transferring the expression plasmid into a HEK293 cell for recombinant expression, after transfecting the cell for 5-6 days, taking a culture supernatant, and purifying the expression supernatant by using a ProA affinity chromatography column to obtain the humanized antibody recombinant protein.

Amino acid sequence of SEQ ID NO.14 hzD2-1

EVQLVESGGGLVQPGGSLRLSCAASGFTVSSDYMGWFRQAPGKEREGVACINTNGGETYHANSVSGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGRTNPDSYGGSRCLLAPEYTYWGQGTLVTVSS

The amino acid sequences of CDRs 1, 2 and 3 of the hzD2-1 antigen complementarity determining region are respectively SEQ ID NO: 16. 17 and 18, underlined (according to the definition of Kabat CDRs).

hZD2-2 amino acid sequence of SEQ ID NO.15

EVQLVESGGGLVQPGGSLRLSCAASGYTASSDYMGWFRQAPGKEREGVACINTNGGETYHANSVSGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGRTNPDSYGGSRCLLAPEYTYWGQGTLVTVSS

The amino acid sequences of CDRs 1, 2 and 3 of hzD2-2 antigen complementarity determining region are respectively SEQ ID NO: 16. 17 and 18, underlined (according to the definition of Kabat CDRs).

Example 8: humanized LILRB4 antibody affinity assays

The antibody affinity was determined by capturing the Fc fragment of the antibody with an Ocet QKe system instrument from Fortebio using an anti-human antibody Fc fragment capture Antibody (AHC) biological probe. The chimeric antibody and its corresponding humanized antibody, control antibody 128, were diluted to 4ug/ml in PBS buffer and passed over the surface of an AHC probe (Cat: 18-0015, PALL) for 120s. LILRB4 recombinant protein (purchased from Yinqiao, cat.16742-H08H) 100nm; the mobile phase had an association time of 300s and a dissociation time of 300s. After the experiment, blank control response values were deducted, and the software was run for 1:1Langmuir binding pattern was fitted and kinetic constants for antigen-antibody binding were calculated.

The kinetic parameters are shown in table 4 below. The results indicate that the affinity of the humanized antibody remains substantially the same as that of the chimeric antibody.

TABLE 4 affinity assay results for humanized antibodies and human LILRB4 recombinant proteins

Sample(s)	KD(M)	kon(1/Ms)	kdis(1/s)
				193	7.21E-10	3.05E+05	2.20E-04
chD2	3.12E-10	2.98E+05	9.30E-05
				hzD2-1	1.03E-09	1.67E+05	1.72E-04
hzD2-2	9.48E-10	1.85E+05	1.75E-04

Example 9: FACS detection of binding Activity of anti-LILRB 4 humanized antibody on RPMI-8226 cells

2E5 RPMI-8226 cells were combined with different concentrations of the anti-LILRB 4 humanized antibody, and hzD2-1 and hzD2-2 humanized antibodies were diluted in 10 gradients starting at 22nM in a 3-fold gradient. Incubated at 4 ℃ in the dark for 60min, washed thoroughly with PBS, then added with FITC-labeled goat anti-human antibody (sigma, F9512) diluted at 1.

The results showed (FIG. 4) that hzD2-1 and hzD2-2 could bind to human LILRB4 positive cells RPMI-8226 cells with binding activity consistent with control antibody 193 and EC50 values are shown in Table 5.

TABLE 5 FACS detection of EC50 values of anti-human LILRB4 humanized antibodies on RPMI-8226 cells

	hzD2-1	hzD2-2	193
				EC50(nM)	0.221	0.215	0.227

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.

Claims (13)

1. A nanobody targeting LILRB4, wherein the nanobody is capable of specifically binding LILRB4, and the CDRs of the VHH chain in the nanobody are one or more selected from the group consisting of:

(1) SEQ ID NO:19, CDR1 shown in SEQ ID NO:20, and SEQ ID NO:21 CDR3;

(2) SEQ ID NO:28, CDR1 shown in SEQ ID NO:29, and the CDR2 shown in SEQ ID NO: CDR3 shown in FIG. 30;

(3) SEQ ID NO:32, CDR1 shown in SEQ ID NO:33, and the CDR2 shown in SEQ ID NO:34 CDR3;

(4) The amino acid sequence of SEQ ID NO:35, SEQ ID NO:36, and the CDR2 shown in SEQ ID NO:37, CDR3 shown.

2. Nanobody according to claim 1, characterized in that it is a humanized VHH or a camelized VH.

3. The nanobody of claim 1, wherein the amino acid sequence of the nanobody has the amino acid sequence as set forth in SEQ ID NO: 4. 7, 9 or 12.

4. A fusion protein comprising a functional domain capable of specifically binding to LILRB4, said functional domain consisting of the nanobody against LILRB4 of any one of claims 1 to 3.

5. An anti-LILRB 4 antibody, wherein the antibody is a conventional antibody or a functional fragment thereof, and the heavy chain variable region of the antibody is composed of nanobody against LILRB4 according to any one of claims 1 to 3.

6. The anti-LILRB 4 antibody of claim 5, wherein said functional fragment is the Fab, fab ', (Fab') 2, fv, scFv or sdFv structure of said classical antibody.

7. A pharmaceutical composition comprising a nanobody against LILRB4 of any one of claims 1 to 3, or a fusion protein of claim 4, or an antibody of claim 5 or 6, and a pharmaceutically acceptable excipient.

8. An isolated nucleic acid molecule encoding the anti-LILRB 4 nanobody of any one of claims 1 to 3, or encoding the fusion protein of claim 4, or encoding the antibody of claim 5 or 6.

9. An expression vector comprising the nucleic acid molecule of claim 8.

10. A recombinant cell comprising the expression vector of claim 9.

11. A method for preparing nanobody according to any one of claims 1 to 3, characterized in that it comprises: culturing the recombinant cell of claim 10, and separating and purifying the nanobody from the culture product.

12. A method of activating a T cell, comprising contacting a T cell with the anti-LILRB 4 nanobody of any one of claims 1 to 3, the fusion protein of claim 4, the antibody of claim 5 or 6, the composition of claim 7, the nucleic acid molecule of claim 8, the vector of claim 9, or the recombinant cell of claim 10, said contacting comprising in the presence of a cancer cell.

13. Use of the anti-LILRB 4 nanobody of any one of claims 1 to 3, the fusion protein of claim 4, the antibody of claim 5 or 6, the composition of claim 7, the nucleic acid molecule of claim 8, the vector of claim 9, or the recombinant cell of claim 10 for the preparation of a medicament for the prevention, treatment and/or amelioration of a tumor.

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