Anti-cd28 nanobodies and uses thereof

Abstract

The application provides anti-CD28 nanobodies and uses thereof, and belongs to the technical field of biotechnology. Specifically disclosed are several CD28-targeted nanobodies, coding sequences and expression vectors for producing the nanobodies, and application of the nanobodies in preparation of tumor treatment drugs. The nanobodies can not only specifically recognize and bind to CD28, laying a foundation for subsequent development of antibody conjugated drugs and multi-specific antibodies, but also can be combined with tumor antigen-targeted antibodies to form multi-specific antibodies, and the formed multi-specific antibodies significantly enhance the ability of activated T cells and the activity of killing tumor cells.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to anti-CD28 nanobodies and their uses. Background Technology

[0002] T cell activation during the immune response requires a dual signal. The first signal comes from the T cell receptor (TCR) recognizing the major histocompatibility (MHC) / antigen peptide complex. The second signal is a co-stimulatory signal provided by CD80 (B7-1) or CD86 (B7-2) on antigen-presenting cells (APCs) stimulating CD28. This dual signaling enables full T cell activation. CD28 is a homodimeric glycoprotein containing 10 glycosylation modification sites, with a molecular weight of 46 kDa, and is expressed on T cells and plasmablasts. CD28 co-stimulatory signaling has multiple biological functions: it promotes T cell proliferation by enhancing the expression of proteins related to T cell proliferation and survival, such as IL2 and IL2R; it enhances T cell survival by expressing Bcl-XL; it produces IL-4 (leading to Th2 differentiation), IFNγ, IL-1, TNF, IL-5, various chemokines and their receptors; CD28 signaling can also induce or upregulate the expression of several other co-stimulatory and regulatory molecules in T cells, including ICOS, 4-1BB and CTLA-4, as well as CD40L, a molecule essential for T cell and B cell interaction; in addition, CD28 signaling can also promote the anti-inflammatory function of regulatory T cells.

[0003] Anti-CD28 antibodies, by binding to CD28 on the cell surface, can act as CD80 or CD86 ligands, enhancing T cell activation. Meanwhile, nanobodies, the smallest naturally occurring functional units of immunoglobulins, have become a current research hotspot due to their advantages such as high affinity, high stability, high solubility, high tissue permeability, and low immunogenicity. Therefore, the development of anti-CD28 nanobodies for therapeutic applications holds potential for treating tumors and autoimmune diseases.

[0004] Several CD28 nanobodies are known in the art, such as the anti-CD28 nanobody mentioned by Shanghai Bio-Tech Co., Ltd. in CN119285780A and the anti-CD28 nanobody developed by Peking University Shenzhen Research Institute in CN114605540B. However, the current variety of anti-CD28 antibodies is limited and cannot effectively meet industry needs, making the development of new anti-CD28 nanobodies crucial. Summary of the Invention

[0005] The purpose of this invention is to provide nanobodies or antigen-binding fragments that specifically bind to CD28, nucleic acids encoding these antibodies and antigen-binding fragments, and uses comprising said antibodies and antigen-binding fragments.

[0006] In some embodiments, the present invention comprises a CD28-specific nanobody or antigen-binding fragment comprising a combination of CDRs, wherein the combination of CDRs comprises CDR1, CDR2 and CDR3.

[0007] CDR1 is selected from SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:71, SEQ ID NO:91, SEQ ID NO:94, SEQ ID NO:97, SEQ ID NO:100, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:108, SEQ ID NO:111, SEQ ID NO:114, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:122, SEQ ID NO:125, SEQ ID NO:91, SEQ ID NO:130, SEQ ID NO:133, SEQ ID NO:83, SEQ ID NO:137, SEQ ID NO:140, SEQ ID NO:143, SEQ ID NO:146, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:152, SEQ ID One of NO:154, SEQ ID NO:156, SEQ ID NO:97, or SEQ ID NO:159;

[0008] CDR2 is selected from one of SEQ ID NO:72, SEQ ID NO:75, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:84, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:112, SEQ ID NO:115, SEQ ID NO:95, SEQ ID NO:120, SEQ ID NO:123, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:131, SEQ ID NO:134, SEQ ID NO:123, SEQ ID NO:138, SEQ ID NO:141, SEQ ID NO:144, SEQ ID NO:147, SEQ ID NO:72, SEQ ID NO:89, SEQ ID NO:144, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:98, SEQ ID NO:112;

[0009] CDR3 is selected from one of SEQ ID NO:73, SEQ ID NO:76, SEQ ID NO:79, SEQ ID NO:82, SEQ ID NO:85, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:93, SEQ ID NO:96, SEQ ID NO:99, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:107, SEQ ID NO:110, SEQ ID NO:113, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:121, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:139, SEQ ID NO:142, SEQ ID NO:145, SEQ ID NO:148, SEQ ID NO:90, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:142, SEQ ID NO:158, SEQ ID NO:99, SEQ ID NO:160.

[0010] Each CDR1, CDR2, and CDR3 is coded according to a traffic analysis method based on KABAT, Chothia, or IMGT; preferably, the traffic analysis method of IMGT is used.

[0011] The CDR1, CDR2, and CDR3 have any combination of sequences selected from the following or have 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to the above-described sequence combinations; preferably, the substitutions are substitutions of conserved amino acids.

[0012] In particular, the nanobodies or antigen-binding fragments of the present invention, wherein the CDR sequences are combined as follows:

[0013] .

[0014] Alternatively, CDR1, CDR2, and CDR3 may be sequences having 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to the above sequence combinations; preferably substitutions, more preferably substitutions of conserved amino acid residues.

[0015] In another specific embodiment, the nanobody or antigen-binding fragment provided by the present invention is characterized in that the nanobody or antigen-binding fragment comprises:

[0016] (i) The variable region has the amino acid sequence shown in SEQ ID NO:1-35;

[0017] (ii) Compared with the amino acid sequence shown in (i), it has one or more amino acid substitutions, deletions, or additions; or

[0018] (iii) The amino acid sequence that has at least 90% sequence identity with the amino acid sequence shown in (i).

[0019] In a preferred embodiment, the nanobody or antigen-binding fragment provided by the present invention constitutes a polypeptide construct that specifically binds to CD28, the polypeptide construct comprising the nanobody antigen-binding fragment described above and an Fc domain, wherein the Fc domain is selected from the sequence of any constant region of antibody IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.

[0020] Preferably, the sequence contains a constant region of human or mouse antibody IgG1, IgG2, IgG3 or IgG4;

[0021] Preferably, the sequence contains the constant region of human IgG4.

[0022] The antibody Fc domain is linked to the antibody or antigen-binding fragment via or without a linker peptide.

[0023] In a preferred embodiment, the antibody or antigen-binding fragment of the present invention is chimeric, humanized, or fully human; preferably, the antibody or antigen-binding fragment is selected from monoclonal antibodies, polyclonal antibodies, natural antibodies, engineered antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, full-length antibodies, antibody fragments, naked antibodies, conjugated antibodies, humanized antibodies, fully human antibodies, Fab, Fab', F(ab')2, Fd, Fv, scFv, diabody, or single-domain antibodies.

[0024] In some embodiments, the present invention provides an isolated nucleic acid molecule, characterized in that the nucleic acid molecule comprises a nucleic acid encoding the nanobody or antigen-binding fragment or the polypeptide construct described above.

[0025] In some embodiments, the present invention provides a recombinant nucleic acid vector, characterized in that the vector comprises the isolated nucleic acid molecules described above.

[0026] In some embodiments, the present invention provides a host cell, characterized in that the host cell contains the isolated nucleic acid molecules or nucleic acid carriers described above.

[0027] In a preferred embodiment, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from mammalian cells, yeast cells, insect cells, Escherichia coli, and / or Bacillus subtilis; even more preferably, the host cell is selected from HEK293E or Chinese hamster ovary cells (CHO).

[0028] In some embodiments, the present invention provides a method for preparing the above-described nanobodies or antigen-binding fragments, or the above-described polypeptide constructs, characterized by comprising culturing the above-described host cells under conditions that allow protein expression, and recovering the nanobodies or their antigen-binding fragments or polypeptide constructs from the cultured host cell culture.

[0029] In a preferred embodiment, the present invention provides a bispecific or multispecific antibody, characterized in that the bispecific or multispecific antibody comprises the nanobody or antigen-binding fragment or the polypeptide construct described above.

[0030] Preferably, the bispecific or multispecific antibody includes at least a first antigen-binding module and a second antigen-binding module. The first antigen-binding module includes the nanobody or antigen-binding fragment as described in claim 1, and the second antigen-binding module specifically binds to antigens other than CD28 or binds to CD28 antigen epitopes different from those of the first antigen-binding module.

[0031] Preferably, the other antigens are selected from DLL3, CD3, PD-1, PD-L1, Her2, EpCAM, CD16, CD20, CD30, CD33, CD47, CD52, CD64, CD133, CEA, gpA33, Mucins, TAG-72, CIX, PSMA, folate-binding protein, GD2, GD3, GM2, VEGF, VEGFR, Integrin, αVβ3, α5β1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, or FAP;

[0032] Preferably, the bispecific or multispecific antibody comprises a first antigen-binding module, a second antigen-binding module, and a third antigen-binding module. The first antigen-binding module comprises the nanobody or antigen-binding fragment described above. The second antigen-binding module specifically binds to the DLL3 antigen epitope, and the third antigen-binding module specifically binds to the CD3 antigen epitope.

[0033] Preferably, the multispecific antibody is "bispecific", "trispecific", or "quadrispecific".

[0034] In a preferred embodiment, the present invention provides a conjugate characterized in that the conjugate comprises the nanobody or its antigen-binding fragment or the polypeptide construct described above, and a therapeutic agent linked to the nanobody or antigen-binding fragment or polypeptide construct described above.

[0035] In a preferred embodiment, the present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises the above-described nanobody or its antigen-binding fragment, the above-described polypeptide construct, the above-described nucleic acid molecule, the above-described recombinant nucleic acid vector, the above-described host cell, the above-described bispecific or multispecific antibody, and the above-described conjugate.

[0036] In a preferred embodiment, the present invention provides the use of the nanobody or its antigen-binding fragment, the polypeptide construct, the nucleic acid molecule, the recombinant nucleic acid vector, the host cell, the bispecific or multispecific antibody, the conjugate, or the pharmaceutical composition for the preparation of a drug. Attached Figure Description

[0037] Figure 1 The results show the detection of the binding of the candidate nanobody to the CD28-HSA antigen and to the CD28 antigen on the cell surface.

[0038] Figure 2 The expression results are for the VHH-Fc chimeric protein composed of the candidate nanobodies.

[0039] Figure 3 shows the affinity results of the VHH-Fc chimeric protein composed of the candidate nanobodies;

[0040] in Figure 3A Affinity curves of the chimeric protein at different concentrations; Figure 3B This is a comparison chart of the affinity results for chimeric proteins.

[0041] Figure 4 The results show the agonistic activity evaluation of the VHH-Fc chimeric protein composed of candidate nanobodies.

[0042] Figure 5 shows the structure of the trispecific antibody constructed from the candidate nanobodies;

[0043] in Figure 5A This is a structural diagram of a trispecific antibody containing the candidate nanobody. Figure 5B and Figure 5C Here is a diagram of the control antibody structure for the example;

[0044] The green part is the DLL3-targeting antibody, the red part is the CD3-targeting antibody, the orange part is the CD28-targeting nanobody, the blue part is the nanobody containing the anti-ALFA tag, and the gray part is the human IgG4 framework region.

[0045] Figure 6 The affinity results for the constructed trispecific antibody are shown.

[0046] Figure 7 The in vitro killing results of the constructed trispecific antibody are shown. Detailed Implementation

[0047] The present invention will now be described in further detail with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the invention. The purpose of providing these embodiments and examples is to enable a more thorough and complete understanding of the disclosure of the present invention. It should also be understood that the present invention can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the invention, and the equivalent forms obtained also fall within the protection scope of this application. Furthermore, numerous specific details are set forth in the following description to provide a more complete understanding of the present invention.

[0048] 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 invention pertains. The terminology used herein in the specification of this invention is for descriptive purposes only and is not intended to limit the invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0049] Terminology Definition

[0050] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0051] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0052] In this invention, terms such as "multiple", "various", "multiple times", and "multi-source" are used, and unless otherwise specified, they refer to a quantity greater than or equal to 2. For example, "one or more" means one or more types.

[0053] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0054] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this invention, solve the technical problem of this invention, and achieve the expected technical effect of this invention.

[0055] In this article, terms such as "preferred," "better," "more suitable," and "ideal" are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this invention.

[0056] In this invention, terms such as "further," "even more," and "particularly" are used for descriptive purposes and to indicate differences in content, but should not be construed as limiting the scope of protection of this invention.

[0057] In this invention, "optionally," "optionally," and "optional" mean that they are optional, that is, they are selected from either "with" or "without." If multiple "options" appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "option" is independent.

[0058] In this invention, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on the quantity.

[0059] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0060] In this invention, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous, and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0061] Unless otherwise specified, the temperature parameters in this invention can be either constant temperature treatment or variations within a certain temperature range. It should be understood that the constant temperature treatment allows temperature fluctuations within the precision range controlled by the instrument. Fluctuations are permitted within ranges such as ±5℃, ±4℃, ±3℃, ±2℃, and ±1℃.

[0062] In this invention, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0063] All references to this invention are incorporated herein by reference as if each document were individually incorporated by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, the referenced documents involved in this invention are incorporated in their entirety and for all purposes. When references are made in this invention, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When references are made in this invention, examples and preferred embodiments of the relevant technical features cited may also be incorporated herein by reference, but only to the extent that they enable the implementation of this invention. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptively based on the description in this application.

[0064] As used in this article, the term “affinity” refers to the strength of the non-covalent interaction between an immunoglobulin molecule (i.e., an antibody) or a fragment thereof and an antigen.

[0065] As used herein, the term "vector" refers to a medium into which a genetic element can be operatively inserted to achieve expression of said genetic element, such as producing a protein, RNA, or DNA encoded by said genetic element, or replicating said genetic element. Vectors can be used to transform, transduce, or transfect host cells to express a carried genetic element within the host cells. Examples of vectors include plasmids; phage particles; granules; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages, such as λ phage or M13 phage; and animal viruses. Vectors may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. Additionally, vectors may contain an origin of replication. Vectors may also include materials that facilitate their entry into cells, including but not limited to viral particles, liposomes, or protein envelopes. Vectors may be expression vectors or cloning vectors. This disclosure provides a nucleic acid sequence containing an antibody or antigen-binding fragment thereof encoded herein, at least one promoter (e.g., SV40, CMV, EF-1α) operatively linked to the nucleic acid sequence, and at least one vector (e.g., an expression vector) containing a selectable marker.

[0066] As used in this article, “host cell” refers to a cell in which exogenous polynucleotides and / or vectors have been introduced.

[0067] As used herein, the term "CD28," an acronym for "differentiation cluster 28," refers to an antigen expressed on T cells as a marker receptor. As used herein, "CD28-related disease" or condition refers to any disease or condition associated with CD28 and / or CD28-expressing cells, such as CD28+ T cells. In some embodiments, CD28-related diseases or conditions are, for example, autoimmune diseases, cancer, adaptive immune diseases, inflammatory diseases, or infectious diseases.

[0068] Those skilled in the art will understand that the CDR region can be determined using any of the Kabat, IMGT, or Chothia antibody numbering systems. Different numbering systems may result in different CDR regions for the same variable region's amino acid sequence. Regardless of the numbering system used, all CDR regions determined fall within the scope of this invention. Unless otherwise stated, the amino acid sequences of specific CDR regions described in this application are determined according to the IMGT antibody numbering system.

[0069] As is well known in the art, the binding specificity and affinity of an antibody are primarily determined by the CDR sequence. Based on mature and well-known existing technologies, the amino acid sequence of non-CDR regions can be easily altered to obtain variants with similar biological activities. Therefore, this application also includes "functional derivatives" of this neutralizing antibody. A "functional derivative" refers to a variant with amino acid substitutions, and a functional derivative retains detectable binding protein activity. A "functional derivative" may comprise both "variants" and "fragments," and because it has the exact same CDR sequence as the neutralizing antibody described in this invention, it possesses similar biological activity.

[0070] The antibodies described herein may contain one or more substituted, deleted, or inserted amino acids relative to their CDR sequence, for example, the number of amino acid insertions, substitutions, or deletions not exceeding three, preferably one. Substitutions, deletions, or insertions can be introduced into the nucleic acid molecule encoding the antibody of the present invention using conventional techniques such as site-directed mutagenesis or PCR-mediated mutagenesis. In some embodiments, conserved amino acid substitutions are performed at one or more sites. "Conserved amino acid substitution" is the case where one amino acid residue is replaced by an amino acid residue having a similar side chain. Amino acid families with similar side chains are defined in the prior art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, aspartamine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). A "variable region" or "variable domain" refers to the amino-terminal domain of the antibody's heavy chain. The variable domain of the heavy chain can be referred to as a "VH". These domains are typically the most variable parts of the antibody and contain antigen-binding sites. The heavy chain variable region (VH) consists of a framework region broken by three hypervariable regions called "complementarity-determining regions" or "CDRs". The structural region of an antibody, which is the combination of the light and heavy chains that make up the antibody, plays a role in positioning and aligning the CDR, which is mainly responsible for binding to the antigen.

[0071] Detailed Implementation Plan

[0072] The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this invention, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0073] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0074] Example 1: Preparation of anti-CD28 nanobody

[0075] 1. Construction of immune Bactrian camel and phage libraries

[0076] A fusion protein (Acro) of the extracellular domain (P10747, Asn19-Pro152) of the human CD28 protein and polyhistidine (PolyHis) was used as an immunogen to immunize Bactrian camels. Healthy adult Bactrian camels were used, and at week 0, immunization was initiated by subcutaneous injection of a mixture of 0.2 mg CD28-PolyHis and complete Freund's adjuvant. Subsequently, booster immunizations were administered every 2 weeks using a mixture of 0.2 mg CD28-PolyHis and incomplete Freund's adjuvant until week 8. Antigen titers were determined by ELISA. Peripheral blood was collected for the construction of a phage display library.

[0077] Peripheral blood lymphocytes (PBMCs) were isolated and purified from whole blood using Ficoll density gradient centrifugation. Total RNA was then extracted using Trizol reagent and reverse transcribed into cDNA using reverse transcription PCR (RT-PCR). The camel heavy chain antibody variable region (VHH) gene was amplified by nested PCR using specific primers.

[0078] The VHH gene fragment was cloned into the pSCD-2 plasmid by BspQI (Hzymes) restriction enzyme digestion and transformed. Escherichia coli Phage display libraries were obtained from strain TG-1, and the library size was subsequently identified, with library diversity assessed by random sequencing. The identified library size was 2.3 × 10⁻⁶. 9 And the quality is up to standard.

[0079] 2. Phage Display Library Selection

[0080] To select nanobodies that effectively bind to CD28, a four-round panning enrichment process was employed, combining cell panning and liquid-phase protein panning. Jurkat cells were used as target cells in the first and third rounds of cell panning. The CD28-HSA fusion protein was used as the antigen in the second and fourth rounds of liquid-phase protein panning. Phages from each round of panning were then infected with TG1 cells and amplified before being used in the next round of enrichment.

[0081] 2.1 Cell Panning Method

[0082] Pre-block 1.5 mL EP tubes with 2% M-PBS and incubate at 37°C for 1 hour. Then, add 3 × 10⁻⁶ ppm of PBS to the blocked EP tubes. 7 One Jurkat cell (1 mL) and 5 μL CD28 phage library (2.65 × 10⁻⁶) 11 PFU), incubate by rotation at room temperature for 1 hour. After centrifugation for 3 minutes, discard the supernatant, wash 3 times with PBS solution, and elute with Trypsin.

[0083] 2.2 Protein Liquid Phase Panning Method

[0084] The protein liquid phase panning was divided into two parts. First, the phage library that did not specifically bind to the magnetic beads was removed. That is, 100 μL of Dynabeads™ MyOne™ Streptavidin T1 magnetic beads and 1 mL of 2% M-PBS were added to the EP tube and the tube was rotated at room temperature and 25 rpm for 1 h to block the phage library. The supernatant was discarded and the phage library diluted with 1% M-PBS was added. The tube was rotated at room temperature and 25 rpm for 1 h to remove the background that bound to the magnetic beads.

[0085] Finally, phage libraries that specifically bind to the antigen were screened. 100 μL of Dynabeads™ MyOne™ Streptavidin T1 magnetic beads and 1 mL of 2% M-PBS were added to EP tubes, and the tubes were blocked by rotation at 25 rpm for 1 h at room temperature. The supernatant was discarded, and 10 μg of biotinylated CD28-HSA fusion protein diluted with PBS and 1 mL of 2% M-PBS were added. The tubes were again blocked by rotation at 25 rpm for 1 h at room temperature. After discarding the supernatant, phage libraries that had not specifically bound to the magnetic beads in the previous step were added, and the tubes were bound by rotation at 25 rpm for 1 h at room temperature. The tubes were washed 15 times with 0.1% PBS-Tween 20, with each wash performed by rotation at 25 rpm for 1 min at room temperature. The tubes were then eluted with 800 μL of 1 mg / mL trypsin, rotated at 25 rpm for 8 min at room temperature, and the reaction was terminated by adding 800 μL of 4% M-PBS.

[0086] 3. Identification of positive clones of anti-CD28 nanobody by ELISA

[0087] First, the enriched phages were selected and single clones were prepared. Then, single clones were picked and inoculated into 100 μL of 2×YT medium containing 50 μg / ml ampicillin, and cultured overnight. Next, helper phage M13KO7 was inoculated at a cell:phage ratio of 1:6, and the culture was incubated at 37°C for 30 min, then at 180 rpm for 30 min. 100 μL of 2×YT medium containing 50 μg / ml ampicillin and 25 μg / ml kanamycin was added, and the culture was incubated overnight at 30°C and 220 rpm. The culture supernatant was then collected by centrifugation and used for single-clone ELISA detection.

[0088] Add 50 μL of human CD28 protein to each well of a 96-well microplate and incubate overnight at 4°C. Wash three times with PBS buffer, add 300 μL of blocking buffer to each well, and incubate at 37°C for 1 hour, discarding the supernatant. Add 300 μL of phage culture supernatant to each well and incubate at 37°C for 1 hour, discarding the supernatant. Then wash three times with PBS buffer, add 50 μL of HRP-labeled anti-M13 antibody to each well, and incubate at 37°C for 1 hour, discarding the supernatant. Wash four times with PBS buffer, add 50 μL of TMB chromogenic buffer to each well, stop the reaction with 2 M H2SO4, and measure the absorbance at 450 nm using a microplate reader. Select clones that bind to CD28 protein for subsequent sequencing identification.

[0089] Example 2: CD28 nanobody sequencing

[0090] 1. Sequencing of positive clones

[0091] Ninety-five positive monoclonal antibodies were selected based on ELISA test data. The bacterial cultures of the positive clones were taken from the monoclonal ELISA test plate, inoculated, and sent to BGI Genomics for sequencing.

[0092] 2. Sequence Analysis

[0093] The sequenced sequences were analyzed using software for sequence alignment, and 35 unique positive clone sequences were selected. The CDR region sequences were then determined according to the IMGT numbering system. The sequence information of the obtained monoclonal nanobodies is shown in Table 1.

[0094] Table 1. Candidate molecular sequences of CD28 nanobodies and their CDR region sequence information.

[0095]

[0096] Example 3: Identification of the binding activity of anti-CD28 chimeric antibody

[0097] 1. Construction and preparation of anti-CD28 recombinant chimeric antibody

[0098] To evaluate the binding ability of CD28 nanobodies to CD28 on the cell membrane surface, a chimeric antibody fused with human IgG1 Fc was further constructed.

[0099] Thirty-five anti-CD28 positive clone sequences obtained from sequencing were cloned into a vector, transiently transfected into HEK293 cells, and cultured at 37°C and 5% CO2 for 48 hours. After culture, the supernatant was collected for subsequent FACS and ELISA identification.

[0100] 2. FACS identification of anti-CD28 nanobodies

[0101] First, add 1×10 to a 1.5 mL centrifuge tube. 6 Jurkat cells were centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. 100 μL of cell expression supernatant was added to an EP tube, along with single-cell and secondary antibody controls, and incubated at room temperature for 1 hour. After washing twice with PBST, Goat pAb to Human IgG (PE) was added, and the reaction was carried out at room temperature in the dark for 1 hour. After washing twice, the cells were resuspended in 300 μL PBS, and finally, FL2 was detected using flow cytometry. The results were analyzed using FlowJo™ 10 software. Results are as follows: Figure 1 As shown.

[0102] 3. ELISA identification of positive clones of anti-CD28 nanobody

[0103] Simultaneously, the binding ability of cell expression supernatant to CD28 antigen was detected using ELISA. CD28-HSA was diluted 1:1 with coating buffer and added to 96-well plates at 100 μL / well, incubated overnight at 4°C. The supernatant was discarded, and the cells were washed three times with PBST. 300 μL of 4% skim milk powder-PBS was added for blocking, and the plates were incubated at 37°C for 1 h. The supernatant was discarded, and the cells were washed three times with PBST. 100 μL of the sample was added, and the control group was added with an equal volume of PBS. The plates were incubated at 37°C for 1 h. The supernatant was discarded, and the cells were washed three times with PBST. HRP-goat anti-human IgG (Fc) was diluted 1:5000 at 100 μL / well, and the plates were incubated at 37°C for 1 h. The supernatant was discarded, and the cells were washed five times with PBST. 100 μL of TMB chromogenic solution was added, and the reaction was developed in the dark. 50 μL of 2M HCl was added to terminate the reaction. The A450 value was measured using a microplate reader.

[0104] The results are as follows Figure 1 As shown, all 35 phage ELISA-positive clones were able to bind to the CD28-HSA antigen after the preparation of cell expression supernatant.

[0105] Example 4: Functional evaluation analysis of anti-CD28 chimeric antibody

[0106] 1. Expression of anti-CD28 chimeric antibody

[0107] To evaluate the affinity and agonistic activity of the CD28 nanobody, we re-prepared the chimeric antibody. HEK293 cells were transiently transfected and cultured for 6 days at 5% CO2. The supernatant was collected, and the antibody was purified by affinity chromatography (Protein A) to obtain homogeneous anti-CD28 VHH-Fc chimeric antibody. Subsequently, the purified antibody was analyzed by SDS-PAGE, and the results are shown below. Figure 2 As shown.

[0108] 2. Affinity of anti-CD28 chimeric antibody

[0109] The binding affinity between CD28 nanobodies and CD28 on the cell membrane was detected using FACS. Two × 10⁻⁶ nanobodies were added sequentially. 5 Jurkat cells and 50 μL of serially diluted chimeric antibody were added to 96-well plates and incubated at 4°C for 2 hours. After washing twice, 100 μL of diluted goat anti-human Fc antibody was added, and the cells were incubated at 4°C for 1 hour. Flow cytometry analysis was then performed. Each CD28 nanobody showed binding ability to CD28 on the cell membrane, with affinity values ​​ranging from single-digit to three-digit nM. Results are as follows: Figure 3A and Figure 3B As shown.

[0110] 3. Evaluation of the agonistic activity of the anti-CD28 recombinant chimeric antibody

[0111] Serially diluted CD28 chimeric antibody and OKT3 antibody (1 μg / mL) were added to 96-well plates at concentrations of 20 μg / mL, 4 μg / mL, 0.8 μg / mL, 0.16 μg / mL, and 0.032 μg / mL, respectively. Anti-ALFA-tagged nanobodies were used as negative controls. The plates were coated overnight at 4°C. The next day, the plates were washed with PBS, and Jurkat (IL2-Luc) cells (Cobioer Biosciences) were added and cultured at 37°C for 6 hours. Finally, cell lysis buffer and luciferase substrate (Cobioer Biosciences) were added, and the luminescence intensity was measured using a microplate reader. Results are shown below. Figure 4 As shown, this indicates that the CD28 chimeric antibody has agonistic activity.

[0112] Example 5: Preparation and Functional Evaluation of Trispecific Antibodies

[0113] 1. Design of trispecific antibodies

[0114] We selected the aforementioned CD28 nanobody sequence, designed and constructed a trispecific antibody (DLL3 / CD28 / CD3), and evaluated its application potential in antitumor immunotherapy.

[0115] An asymmetric trispecific antibody was designed, with the molecular form shown in Figure 5. The trispecific antibody consists of two chains: Chain A: DLL3 ScFv-CD3 ScFv-Fc1; Chain B: CD28 VHH-Fc2. A knob-in-hole design was used to form a heterodimer of the heavy chain. The Fc sequence was derived from the constant region of the heavy chain of human IgG4. Simultaneously, amino acid mutations were introduced at F234A and L235A (according to Kabat's "EU" designation) to reduce the binding affinity to the Fcγ receptor.

[0116] The control antibodies were set up as follows: AMG757 (Tarlatamb, KEGG#D12234), AMG757-KIH, and TSAb-ALFANB. AMG757-KIH does not contain anti-CD28 VHH, and TSAb-ALFANB is a DLL3 / ALFA / CD3 trispecific antibody.

[0117] Prepare trispecific antibodies according to the combinations in Table 2.

[0118] Table 2. List of trispecific antibodies and their control sequences

[0119]

[0120] 2. Expression and purification of trispecific antibodies

[0121] The sequences in Table 2 were synthesized into pCDNA 3.1 and transiently expressed in HEK293 cells. The two plasmids expressing the two strands were co-transfected at a 1:1 ratio. After 6 days of expression, the cell supernatant was harvested. Homogeneous antibody molecules were obtained by purification and separation using Protein A affinity chromatography and cation exchange chromatography. Purity was identified using SEC-HPLC, and the purity of each antibody was higher than 95%. These antibodies were used in the following examples.

[0122] 3. Assess the binding ability of trispecific antibodies to antigens.

[0123] The binding ability of antibody molecules was evaluated using the FACS method as described in Example 3.

[0124] The results are as follows Figure 6 As shown, the antibody molecules prepared in Example 5 all possess the ability to bind to cell membranes and express antigens. However, compared to AMG757, the binding ability of AMG757-KIHFc and TSAb-ALFANB to Jurkat cells was slightly reduced, possibly due to differences in affinity between the secondary antibodies and different antibody Fc subtypes. The binding ability of the trispecific antibody molecules prepared in this example to Jurkat cells was enhanced, indicating that the anti-CD28 nanobody element in the trispecific antibody performed its binding function.

[0125] 4. Evaluate the ability of trispecific antibodies to kill tumors in vitro.

[0126] CD3 / CD28 / DLL3 trispecific antibodies can bind to CD3 and CD28 molecules on the surface of T cells, and simultaneously bind to DLL3 molecules on the surface of tumor cells, bridging T cells and tumor cells and mediating T cell killing of tumor cells.

[0127] In this embodiment, SHP77 cells (Luc) labeled with CFSE dye (ThermoFisher) were used as target cells, and PBMCs were used as effector cells to evaluate the TDCC activity mediated by the prepared trispecific antibody molecules. 5 × 10⁵ cells were added to 96-well plates sequentially. 4 2.5 × 10⁶ SHP-77 cells (pre-labeled with CFSE dye) 5 PBMCs from healthy donors were collected, and then serially diluted antibodies were added. The mixture was incubated at 37°C with 5% CO2 for 7 days. Cell integrity was assessed by staining with 7-AAD (ThermoFisher) dye. Samples were then measured using a CytoFLEX instrument (Beckman) and analyzed using CytExpert software (Beckman). Results are as follows: Figure 7 As shown in Table 3.

[0128] The results showed that the trispecific antibodies prepared in this embodiment possess the ability to kill tumor cells in vitro. Compared with bispecific antibodies against DLL3 and CD3, the CD3 / CD28 / DLL3 trispecific antibodies were able to mediate the killing of tumor cells by PBMCs at lower doses, especially TSAb-Nb7, TSAb-Nb36, TSAb-Nb38, TSAb-Nb73, and TSAb-Nb75.

[0129] Table 3. Results of in vitro killing ability detection of trispecific antibodies

[0130]

Claims

1. A Nanobody or antigen binding fragment that specifically binds to CD28, characterized in that, The complementarity-determining region (CDR) of the nanobody or antigen-binding fragment is composed of (i) Composed of CDR1 shown in SEQ ID NO: 77, CDR2 shown in SEQ ID NO: 78, and CDR3 shown in SEQ ID NO: 79; (ii) Composed of CDR1 shown in SEQ ID NO: 91, CDR2 shown in SEQ ID NO: 128, and CDR3 shown in SEQ ID NO: 129; (iii) Composed of CDR1 shown in SEQ ID NO: 108, CDR2 shown in SEQ ID NO: 109, and CDR3 shown in SEQ ID NO: 110; (iv) Composed of CDR1 shown in SEQ ID NO: 111, CDR2 shown in SEQ ID NO: 112, and CDR3 shown in SEQ ID NO: 113; (v) Composed of CDR1 shown in SEQ ID NO: 114, CDR2 shown in SEQ ID NO: 115, and CDR3 shown in SEQ ID NO: 116; (vi) Composed of CDR1 shown in SEQ ID NO: 137, CDR2 shown in SEQ ID NO: 138, and CDR3 shown in SEQ ID NO: 139; (vii) Composed of CDR1 shown in SEQ ID NO: 140, CDR2 shown in SEQ ID NO: 141, and CDR3 shown in SEQ ID NO:

142.

2. The Nanobody or antigen-binding fragment of claim 1, characterized in that, The variable region amino acid sequence of the nanobody or antigen-binding fragment is SEQ ID NO:3, 21, 14, 15, 16, 25, 26.

3. A polypeptide construct that specifically binds to CD28, characterized in that, The polypeptide construct comprises the nanobody and Fc domain as described in claim 1, wherein the Fc domain is selected from the sequence of any constant region of antibody IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.

4. An isolated nucleic acid molecule, comprising, The nucleic acid molecule contains a nucleic acid encoding the nanobody or antigen-binding fragment of claim 1 or the polypeptide construct of claim 3.

5. A recombinant nucleic acid vector, characterized in that, The carrier includes the nucleic acid molecule as described in claim 4.

6. A host cell, characterized in that, The host cell contains the nucleic acid molecule as described in claim 4.

7. The host cell of claim 6, wherein, The host cells are HEK293 or Chinese hamster ovary cells (CHO).

8. A method of producing the nanobody or antigen binding fragment of claim 1 or the polypeptide construct of claim 3, characterized in that, Including steps, (i) Culturing the host cells of claim 6 under conditions suitable for antibody production; and (ii) Isolate and recover the nanobody or its antigen-binding fragment or polypeptide construct from the cultured host cell culture.

9. A bispecific antibody, characterized in that The bispecific antibody comprises a first antigen-binding module and a second antigen-binding module. The first antigen-binding module comprises the nanobody or antigen-binding fragment as described in claim 1. The second antigen-binding module specifically binds to antigens other than CD28 or binds to different CD28 antigenic epitopes of the first antigen-binding module.

10. A trispecific antibody, characterized in that, The trispecific antibody comprises a first antigen-binding module, a second antigen-binding module, and a third antigen-binding module. The first antigen-binding module comprises the nanobody or antigen-binding fragment as described in claim 1. The second antigen-binding module specifically binds to the DLL3 antigen epitope, and the third antigen-binding module specifically binds to the CD3 antigen epitope.

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