Anti-CD28 antibodies

Abstract

The present application relates to the diagnosis and treatment of diseases, including cancer, chronic infectious diseases, autoimmune diseases, inflammatory conditions, and the prevention of transplant rejection. The present invention provides and relates to the use of antibody molecules that bind to CD28 and also bind to CTLA-4 in a non-hyper-agonistic manner. The antibody molecules may form part of a bispecific molecule that binds, for example, tumor associated antigens or other T cell antigens, such as CD3.

Description

[0001] This application claims the priority of European patent application No. 22186570.2 filed on July 22, 2022 and European patent application No. 23174908.6 filed on May 23, 2023, the contents and elements of which are incorporated herein by reference for all purposes. Field of the Invention

[0002] The present application relates to the diagnosis and treatment of diseases, including cancer, chronic infectious diseases, autoimmune diseases, inflammatory conditions, and the prevention of transplant rejection. The present invention provides and relates to the use of antibody molecules that bind to CD28 from humans and mice in a non-superagonistic manner and also bind to CTLA-4. The antibody molecules may form part of a bispecific molecule that additionally binds to a tumor-associated antigen or other T cell antigen. Background of the Invention

[0004] CD28 is a homodimeric glycoprotein molecule that is expressed on most T cells (approximately 95% of CD4 + and 50% CD8 + CD28 is expressed constitutively on the surface of T cells (Damle et al., 1983). The main function of CD28 is to provide key co-stimulatory signals that enhance T cell proliferation, survival, and the production of key cytokines such as interleukin 2, interferon-γ, and TNF-α, as described below (Jenkins et al., 1991; June et al., 1987; Martin et al., 1986; Weiss et al., 1986).

[0005] T cell activation is defined using a two-signal model (Mueller et al., 1989; Jenkins et al., 1991; Bretscher and Cohn, 1970). Signal one is regulated by T cell receptor (TCR) / CD3 complex engagement (Allison et al., 1982; Hedrick et al., 1984; Yanagi et al., 1984). TCR engagement is induced by recognition of antigenic peptides presented by major histocompatibility complex (pMHC) on antigen presenting cells (APCs) such as B cells (La Gruta et al., 2018; Davis and Bjorkman, 1988). The TCR itself lacks an intracellular signaling domain, and therefore binding to CD3 and other co-receptors such as CD4 and CD8 is essential for the generation of activation signals (Weiss and Stobo, 1984; Ohashi et al., 1985; Weiss et al., 1986; Janeway, 1988). However, the TCR / CD3 complex alone is not sufficient to fully activate T cells. Indeed, the absence of additional signals induces T cell exhaustion, thereby impairing T cell activation (Mueller et al., 1989).

[0006] Signal two is directed by the engagement of co-stimulatory receptors found on the surface membrane of T cells with counter-receptors on APCs (Mueller et al., 1989; Jenkins et al., 1991; Bretscher and Cohn, 1970). CD28 is an example of such a co-stimulatory receptor and binds to its counter-receptors CD80 and CD86 on APCs to provide co-stimulation (Freeman et al., 1989; Freeman et al., 1993(a); Freeman et al., 1993(b); Azuma et al., 1993; Caux et al., 1994).

[0007] In addition to CD28, CTLA-4 is the second ligand of CD80 / CD86. +It is upregulated in T cells and functions as an immune checkpoint by providing inhibitory signals (Brunet et al., 1987). Blocking CTLA-4 using monoclonal antibodies (mAbs) has reduced this inhibitory signal, which has revolutionized the field of cancer immunotherapy (Krummel and Allison, 1995; Leach et al., 1996; Kwon et al., 1997). It has been shown that CTLA-4 shares significant structural homology with CD28, and it outperforms the affinity of CD28 for CD80 / CD86 binding with at least 20 times higher affinity (Linsley et al., 1991; Peach et al., 1994). The CD28 / CTLA-4 pathway is a primitive co-signaling pathway in T cells, and the CTLA-4 co-inhibition acts as a counter-signal to the co-stimulatory effect of CD28 because they bind to the same receptors (CD80 and CD86). Since CD28 co-stimulation is essential for T cell activation, immunomodulation by blocking this pathway using antagonist anti-CD28 antibodies is a promising approach to prevent inappropriate T cell activation in transplant settings and to potentially treat T cell-mediated autoimmune diseases (Crepeau and Ford, 2017).

[0008] Due to its role in T cell activation, CD28 has been identified as a therapeutic target, for example in cancer therapy. Many antibodies that bind to human CD28 have been described in the art. These antibodies can be broadly classified as conventional (agonist) anti-CD28 antibodies or unconventional (superagonist) anti-CD28 antibodies (Tacke et al., 1997; Lühder et al., 2003).

[0009] The combination of agonist antibodies against both the TCR / CD3 complex and CD28 is sufficient to fully activate T cells by cross-linking, so they can replace MHC and CD80 / 86 signaling, respectively. Conventional anti-CD28 mAbs bind close to the natural binding site of CD80 / CD86 and provide co-stimulation only in the presence of TCR / CD3 signaling (Lühder et al., 2003).

[0010] In contrast, superagonist anti-CD28 mAbs bind to the exposed C"D loops of CD28 and fully activate T cells without the need for engagement of the TCR / CD3 complex (Lühder et al., 2003; Tacke et al., 1997). In rat models, this unusual class of antibodies induces potent proliferation of T cells without overt toxicity (Tacke et al., 1997; Rodríguez-Palmero et al., 1999). At low doses, only regulatory T cells (Tregs) are activated, whereas at high doses, both regulatory T cells and conventional T cells are expanded, suggesting that there is preferential activation of T cells at different doses (Lin et al., 2003; Beyersdorf et al., 2005).

[0011] As a result, a humanized anti-CD28 antibody (TGN1412) in the form of IgG4 was developed that binds to human and cynomolgus CD28 for different therapeutic indications (Lühder et al., 2003). In cynomolgus monkeys, TGN1412 was well tolerated at doses up to 50 mg / kg, so an initial dose of 0.1 mg / kg was considered safe for human treatment (Pallardy and Hünig, 2010; Hanke, 2006). However, in 2006, a phase I clinical trial was conducted in which all six volunteers who received TGN1412 developed life-threatening cytokine release syndrome (CRS) with multiple organ failure, which led to the termination of the clinical trial (Suntharalingam et al., 2006).

[0012] The failure to predict the toxicity of TGN1412 in the preclinical phase was due to a number of factors. First, the addition of TGN1412 to human PBMCs in vitro did not induce cytokine release unless these cells were artificially fixed to cell culture wells (Stebbings et al., 2007). Second, a subset of T cells (known as tissue-resident CD4 effector memory cells; CD4EM) is the main source of CRS. Therefore, no toxicity was detected in young and pure laboratory rats that lack this type of cells. Cytokine release in rodents is also quenched by regulatory T cells (Treg cells), which are maintained active by IL-2 produced by conventional T cells ( et al., 2011; Eastwood et al., 2010). Finally, and most importantly, CD4EM cells in cynomolgus monkeys were found to downregulate CD28, and therefore, toxicity in humans cannot be predicted by testing in these non-human primates (Eastwood et al., 2010).

[0013] Currently, no anti-CD28 antibodies have been approved for cancer treatment in human patients. Therefore, there is still a need in the art to further develop anti-CD28 antibodies with a more favorable toxicity profile than TGN1412, for example, for cancer treatment.

[0014] In view of the above considerations, the present invention has been conceived. SUMMARY OF THE INVENTION

[0016] The inventors of the present application have developed human antibody molecules that bind to human CD28 and provide strong co-stimulatory signals, but the antibody molecules do not activate T cells in the absence of TCR / CD3 engagement (i.e., these antibodies are not superagonists). This is in contrast to the known anti-CD28 antibody TGN1412, because activation and proliferation of T cells were observed in the absence of TCR / CD3 engagement (Example 4). Preferably, the antibodies of the present invention are agonistic anti-CD28 antibodies.

[0017] The strong co-stimulatory signal of the antibodies of the invention and the absence of super-agonistic activity are expected to translate into a more favorable toxicity profile than that observed with TGN1412, while enhancing the immune system to fight cancer or chronic infectious diseases. Advantageously, the co-stimulatory signal provided by the antibodies of the invention is stronger than that provided by TGN1412 (Example 3).

[0018] In addition, the antibodies of the present invention also bind to CTLA-4 (Example 5). Both CTLA-4 and CD28 share a conserved binding motif (MYPPPY), which is essential for binding to CD80 / CD86 (Peach et al., 1994). Unlike TGN1412, the antibodies of the present invention bind to an epitope of CD28 that contains a MYPPY binding motif (Example 6).

[0019] Cross-reactivity with CTLA-4 means that the antibodies of the present invention have the potential to act as checkpoint inhibitors in addition to their co-stimulatory activity, further enhancing anti-cancer immune responses. It is believed that the cross-reactivity property with CTLA-4 is not an inherent feature of all non-super-agonist anti-CD28 antibodies, for example, the anti-CD28 antibodies disclosed in US20190389951A1 do not show such properties.

[0020] The antibodies of the present invention also cross-react with murine CD28. Cross-reaction with murine CD28 provides a means for evaluating the efficacy of anti-CD28 antibody molecules. Due to similarities with humans in anatomy, physiology, and genetics, mice represent a useful animal model for evaluating anti-CD28 antibodies and other therapeutic agents. The advantages of mice include their small size, ease of maintenance, short life cycle, and abundant genetic resources, which means that mice provide a promising animal model for translational research to determine the efficacy of anti-CD28 therapeutic agents.

[0021] In a first aspect, the present invention therefore relates to an antibody molecule that binds to CD28. Said antibody molecule preferably comprises the HCDR1, HCDR2 and HCDR3 sequences of the "AE2P" antibody as shown in SEQ ID NOs 3, 4 and 5, respectively, and / or the LCDR1, LCDR2 and LCDR3 sequences of the AE2P antibody as shown in SEQ ID NOs 6, 7 and 8, respectively. In a preferred embodiment, the antibody molecule comprises the VH domain sequence or the VL domain sequence of the AE2P antibody molecule as shown in SEQ ID NOs 9 and 10, respectively, but preferably comprises said VH domain sequence and VL domain sequence. The VH and VL domains of the antibody molecule may be connected by a linker, such as the linker shown in SEQ ID NO 12.

[0022] The antibody molecules of the present invention preferably bind to human CD28, and CD28 from mouse (Mus musculus) (referred to herein as "murine" CD28). The sequence of the extracellular domain of human CD28 is shown in SEQ ID NO: 1, and the sequence of the extracellular domain of mouse CD28 is shown in SEQ ID NO: 2.

[0023] The antibody molecules of the present invention are preferably human antibodies or humanized antibodies. Most preferably, the antibodies of the present invention are fully human antibodies. Fully human antibodies have advantages due to their lower potential for immunogenicity.

[0024] As mentioned herein, the antibody molecule can take any suitable form. In some embodiments, the antibody can bind to CD28 monovalently. In some embodiments, the antibody can bind to CD28 bivalently. Many antibody molecule forms are known in the art, and include both complete antibody molecules, such as IgG, and antibody molecule fragments, such as single-chain Fv (scFv) or single-chain diabodies (scDb). The term "antibody molecule" as used herein covers complete antibody molecules and antibody molecule fragments, particularly antigen binding fragments. Preferably, the antibody molecule comprises a VH domain and a VL domain. In a preferred embodiment, the antibody molecule is or comprises scFv, a small immune protein (SIP), a diabody (Db), a single-chain diabody (scDb), or a (complete) IgG molecule, such as an IgG1, IgG2a or IgG4 molecule. The sequence of the AE2P antibody in the form of a single-chain Fv (scFv) is shown in SEQ ID NO: 11 and 25. The sequence of the AE2P antibody in the form of a diabody (Db) is shown in SEQ ID NO: 19. The sequence of the AE2P antibody in the form of a single-chain diabody (scDb) is shown in SEQ ID NO:65; alternatively, the AE2P antibody in the form of a single-chain diabody (scDb) may have a sequence as shown in SEQ ID NO:20. The sequence of the AE2P antibody in the form of a small immune protein (SIP) is shown in SEQ ID NO:15. The sequence of the AE2P light chain is shown in SEQ ID NO:14, and the sequence of the AE2P heavy chain in the form of IgG1 is shown in SEQ ID NO:13, IgG 2A The sequence of the AE2P heavy chain in the IgG4 format is shown in SEQ ID NO: 17, and the sequence of the AE2P heavy chain in the IgG4 format is shown in SEQ ID NO: 22.

[0025] The antibody molecule of the present invention can be used as is, or can be conjugated with other molecules to provide a conjugate. The selection of the molecule conjugated with the antibody molecule will depend on the intended application of the conjugate. For example, if the conjugate is intended to be used for treating a disease or condition, the conjugate may include the antibody molecule of the present invention and a bioactive agent. The bioactive agent may be a proinflammatory agent. If the conjugate is intended for imaging, detecting or diagnosing a disease or condition, the conjugate may include the antibody molecule of the present invention and a detectable label or marker molecule, such as a radioisotope, such as a non-therapeutic radioisotope. Depending on the molecule conjugated with the antibody molecule, the conjugate may be or may include a single-chain protein. If the conjugate is a single-chain protein, the entire protein may be expressed as a single polypeptide or fusion protein. In this case, the molecule may be conjugated with the antibody molecule by a peptide linker. Fusion proteins have the advantage of being easy to produce and purify, because they are composed of only a single substance, which facilitates the production of clinical-grade materials. Alternatively, the molecule may be conjugated with the antibody molecule by a cleavable linker.

[0026] The antibody molecules of the present invention may constitute part of a bispecific binding molecule. In some embodiments, the bispecific binding molecule comprises an antigen binding site that binds to a tumor-associated antigen, such as fibroblast activation protein (FAP), a splicing isoform of fibronectin, such as ED-A, ED-B or IIICS isoforms of fibronectin, carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), mucin-16, prostate-specific membrane antigen (PSMA) or a splicing isoform of tenascin-C, such as A1, A2, B, C or D isoforms of tenascin-C. In some embodiments, the bispecific binding molecule comprises an antigen binding site that binds to a second T cell antigen such as CD3. The bispecific binding molecules used according to the present invention include bispecific antibodies and may be selected from IgG-added antibodies with additional antigen binding portions (e.g., IgG-(scFv)2 and IgG-(scFv)) or small recombinant bispecific antibody formats (e.g., bispecific T cell engagers (BiTE TM ) and scDb-scFv), or any other molecular format, which comprises a binding molecule specific for a given target and which is coupled to one or two different binding molecules specific for one or two different targets. Examples of bispecific binding molecules can be found in Kontermann 2012 (page 186) Figure 2 ), the contents of which are incorporated herein by reference. Bispecific T cell engagers (BiTE TM The sequences of the anti-human CEA antibody and the anti-FAP antibody 7NP2 in IgG-(scFv)2 format are shown in SEQ ID NO: 23. The heavy chain and light chain sequences of the anti-human CEA antibody and the AE2P antibody in IgG-(scFv)2 format, wherein the anti-human CEA antibody is in IgG format and the AE2P antibody is in scFv format (2+2; format 1, Fig.13 ), as shown in SEQ ID NO: 61 and 62, respectively. The heavy chain and light chain sequences of AE2P antibody and anti-human CEA antibody in IgG-(scFv)2 format, wherein the AE2P antibody is in IgG format and the anti-human CEA antibody is in scFv format (2+2; format 2, Fig.13 ), as shown in SEQ ID NO:63 and 64, respectively.

[0027] The present invention also provides isolated nucleic acids encoding the antibody molecules and conjugates of the present invention. Those skilled in the art will have no difficulty in preparing such nucleic acids using methods well known in the art. Isolated nucleic acids can be used to express the antibody molecules or conjugates of the present invention, for example, by expressing them in bacteria, yeast, insect or mammalian host cells. Preferred host cells are Escherichia coli (E. coli) and CHO-S cells. Typically, nucleic acids will be provided in the form of recombinant expression vectors for expression. In vitro host cells containing such nucleic acids and expression vectors are part of the present invention, and their use for expressing the antibody molecules and conjugates of the present invention is also part of the present invention, and these antibody molecules and conjugates can be subsequently purified from cell cultures and optionally formulated into pharmaceutical compositions.

[0028] The antibody molecule or conjugate of the present invention can be provided, for example, in a pharmaceutical composition, and can be used for medical purposes alone or in combination with one or more other therapeutic agents as described herein. For example, the antibody molecule or conjugate of the present invention can be used in combination with a CD3 agonist (e.g., an antibody binding to human CD3) for medical purposes as described herein. Other exemplary therapeutic agents that can be used in combination with the antibody molecule of the present invention or administered in association therewith include, for example, chemotherapy (e.g., anti-cancer chemotherapy, such as paclitaxel, docetaxel, vincristine, cisplatin, carboplatin or oxaliplatin); radiotherapy; checkpoint inhibitors, such as checkpoint inhibitors targeting PD-1 (e.g., anti-PD-1 antibodies, such as pembrolizumab, nivolumab or cemiplimab (see U.S. Patent No. 9,987,500)), checkpoint inhibitors targeting CTLA-4, LAG3 or TIM3; costimulatory agonist antibodies targeting, for example, GITR, OX40 or 4-1BB; or a second anti-CD28 antibody, such as a second costimulatory CD28 bispecific antibody.

[0029] Alternatively, the antibody molecules or conjugates of the invention may be provided in a diagnostic composition and used for diagnostic purposes as described herein.

[0030] The present invention also relates to an antibody molecule or conjugate of the present invention for use in a method for treating a human or animal body by therapy. For example, the antibody molecule or conjugate of the present invention can be used in a method for treating a patient's cancer, chronic infectious disease, autoimmune disease and / or inflammatory disorder, or in a method for preventing transplant rejection in a patient.

[0031] The present invention also relates to a method for treating cancer, chronic infectious diseases, autoimmune diseases and / or inflammatory conditions in a patient, or a method for preventing transplant rejection, the method comprising administering a therapeutically effective amount of the antibody molecule or conjugate of the present invention to the patient. It is also contemplated that the antibody molecule or conjugate of the present invention is used to prepare a drug for treating cancer, chronic infectious diseases, autoimmune diseases and / or inflammatory conditions in a patient, or to prepare a drug for preventing transplant rejection. Examples of chronic infectious diseases as mentioned herein include chronic hepatitis B infection (HBV), human immunodeficiency virus (HIV) infection, and tuberculosis. Examples of autoimmune diseases that can be treated with the antibody molecule or conjugate of the present invention include lupus erythematosus, rheumatoid arthritis, and psoriatic arthritis. Inflammatory or autoimmune diseases that can be treated with the antibody molecule or conjugate of the present invention include inflammatory bowel disease (IBD), such as Crohn's disease or ulcerative colitis.

[0032] In a preferred embodiment, the antibody molecules or conjugates of the invention are for use in a method for treating cancer.

[0033] Another aspect of the present invention relates to an antibody molecule or conjugate of the present invention for use in a method for imaging, detecting or diagnosing a patient's cancer, chronic infectious disease, autoimmune disease and / or inflammatory condition. The present invention further relates to a method for imaging, detecting or diagnosing a patient's cancer, chronic infectious disease, autoimmune disease and / or inflammatory condition, the method comprising administering an antibody molecule or conjugate of the present invention to the patient. The method may be an in vitro method or an in vivo method. The scope of the present invention also encompasses the use of an antibody molecule or conjugate of the present invention for preparing a diagnostic product for imaging, detecting or diagnosing cancer, chronic infectious disease, autoimmune disease and / or inflammatory condition.

[0034] In a preferred embodiment, the antibody molecules or conjugates of the invention are for use in a method of imaging, detecting or diagnosing cancer.

[0035] The patient as referred to herein is preferably a human patient.

[0036] The present invention includes any combination of the above-mentioned aspects and preferred features, unless such a combination is obviously not allowed or has been explicitly excluded. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Embodiments and experiments illustrating the principles of the present invention will now be discussed with reference to the accompanying drawings, in which:

[0039] Figure 1 Biochemical characterization of AE2P in scFv format is shown. Figure 1A shows the results of size exclusion chromatography of scFv (AE2P). As expected, the monomeric form of scFv was eluted from the S75i GL column at 11.5 mL. Figure 1 B shows the results of SDS-PAGE analysis of scFv (AE2P). The scFv has the expected size of 25 kDa under non-reducing (NR) and reducing (R) conditions, respectively. Figure 1 C shows the titration ELISA of scFv (AE2P) against human CD28-Fc fusion protein. The data showed an EC of approximately 31 nM 50 , confirming the binding of AE2P to CD28.

[0040] Figure 2 Biochemical characterization of the IgG form of AE2P is presented. Figure 2 A shows the results of size exclusion chromatography of IgG4 (AE2P). As expected, IgG4 was eluted from the S200i 10 / 300GL column at 11.9 mL. Figure 2 B shows the results of SDS-PAGE analysis of IgG4 (AE2P). Under non-reducing conditions, IgG4 has the expected size of 150 kDa, and under reducing conditions, it has sizes of 25 kDa and 50 kDa. Figure 2 C shows flow cytometry analysis of AE2P IgG4 and TGN1412 IgG4 (also referred to as TGN) binding to primary human T cells. The dashed line represents the isotype negative control, while the solid fill represents the shift in fluorescence upon binding. This confirms that both AE2P IgG4 and TGN1412 IgG4 bind to primary human T cells expressing CD28.

[0041] Figure 3 Flow cytometry analysis of AE2P IgG4 against various negative cell lines is shown. The dashed line represents the isotype negative control, while the solid line represents AE2P IgG4. This confirms that AE2P IgG4 specifically binds to primary human T cells and not to other negative cell lines that do not express CD28.

[0042] Figure 4 Proliferation of human PBMCs after 3 days of incubation with OKT3 alone or in combination with AE2P IgG4, TGN1412 IgG4 (also referred to as TGN) or isotype IgG4 is shown. This demonstrates the co-stimulatory effect of anti-CD28 antibodies (AE2P IgG4 and TGN1412 IgG4) in combination with anti-CD3 (OKT3) on the proliferation of human PBMCs. The data also show that AE2P IgG4 provides a greater co-stimulatory effect on the proliferation of human PBMCs than TGN1412 IgG4.

[0043] Figure 5 Shown are cytokine release from human PBMCs after 3 days of incubation with OKT3 alone or in combination with AE2P IgG4, TGN1412 IgG4 (also referred to as TGN), or isotype IgG4. Figure 5 A shows the cytokine release of human IL-2. This demonstrates that IL-2 secretion by human PBMCs is increased when anti-CD28 antibodies (AE2P IgG4 and TGN1412 IgG4) are used in combination with anti-CD3 (OKT3). Figure 5 B shows the cytokine release of human interferon-γ. This demonstrates that interferon-γ secretion by human PBMCs is increased when anti-CD28 antibodies (AE2P IgG4 and TGN1412 IgG4) are used in combination with anti-CD3 (OKT3). Figure 5 C shows the cytokine release of human TNF-α. This demonstrates that when anti-CD28 antibodies (AE2P IgG4 and TGN1412 IgG4) are used in combination with anti-CD3 (OKT3), TNF-α secretion by human PBMCs is increased. The data also show that AE2P IgG4 provides greater co-stimulatory effects on the release of all three cytokines compared to TGN1412 IgG4.

[0044] Figure 6 Shown is the proliferation of human PBMCs after incubation with AE2P IgG4, TGN1412 IgG4 (also referred to as TGN), or isotype IgG4 in the absence of OKT3. Figure 6 A shows the proliferation of human PBMCs after 3 days. This demonstrates the lack of proliferation of human PBMCs after 3 days of incubation with AE2P IgG4 alone, unlike TGN1412 IgG4. Figure 6 B shows the proliferation of human PBMCs after 5 days. This demonstrates the lack of proliferation of human PBMCs after 5 days of incubation with AE2P IgG4 alone, unlike TGN1412 IgG4.

[0045] Figure 7 Shown are cytokine release from human PBMCs following incubation with either AE2P IgG4, TGN1412 IgG4 (also referred to as TGN), or isotype IgG4 in the absence of OKT3. Figure 7 A shows the cytokine release of human IL-2. This demonstrates the lack of IL-2 secretion by human PBMCs after 3 days of incubation with AE2P IgG4 alone, unlike TGN1412 IgG4. Figure 7 B shows the cytokine release of human IFN-γ. This demonstrates the lack of IFN-γ secretion by human PBMCs after 3 days of incubation with AE2P IgG4 alone, unlike TGN1412 IgG4. Figure 7C shows the cytokine release of human TNF-α. This demonstrates the lack of TNF-α secretion by human PBMCs after 3 days of incubation with AE2P IgG4 alone, unlike TGN1412 IgG4.

[0046] Figure 8 Shown is the upregulation of activation markers on human PBMCs after 3 days of incubation with AE2P IgG4, TGN1412 IgG4 (also referred to as TGN), or isotype IgG4 in the absence of OKT3. Figure 8 A shows the upregulation of CD69 on CD4-positive T cells. This demonstrates the lack of upregulation of CD69 on CD4-positive T cells in human PBMCs after 3 days of incubation with AE2P IgG4 alone, unlike TGN1412 IgG4. Figure 8 B shows upregulation of CD69 on CD8-positive T cells. This demonstrates the lack of upregulation of CD69 on CD8-positive T cells in human PBMCs after 3 days of incubation with AE2P IgG4 alone, unlike TGN1412 IgG4. Figure 8 C shows upregulation of CD25 on CD4-positive T cells. This demonstrates the lack of upregulation of CD25 on CD4-positive T cells in human PBMCs after 3 days of incubation with AE2P IgG4 alone, unlike TGN1412 IgG4. Figure 8 D shows upregulation of CD25 on CD8-positive T cells. This demonstrates the lack of upregulation of CD25 on CD8-positive T cells in human PBMCs after 3 days of incubation with AE2P IgG4 alone, unlike TGN1412 IgG4.

[0047] Fig. 9 Binding of AE2P to human and mouse CD28 Fc is demonstrated. Fig. 9 A shows the binding of AE2P IgG4 to human CD28 Fc compared to non-binding proteins by titration ELISA. Fig. 9 B shows the binding of AE2P IgG4 to murine CD28 Fc compared to non-binding proteins by titration ELISA. Fig. 9 C shows the binding of TGN1412 (also referred to as TGN) to human CD28 Fc compared to non-binding proteins by titration ELISA. Fig. 9 D shows the lack of binding of TGN1412 (also referred to as TGN) to murine CD28 Fc by titration ELISA compared to its binding to non-binding proteins. Fig. 9E shows the binding of AE2P or TGN1412 to primary mouse T cells as determined by flow cytometry. The dashed line represents the isotype negative control, while the solid line represents AE2P or TGN1412. The results show that AE2P IgG4 binds to primary mouse T cells, while TGN1412 does not.

[0048] Fig.10 Binding of AE2P or TGN1412 (also referred to as TGN) to human CTLA-4 by titration ELISA is shown. This demonstrates that AE2P IgG4 binds to human CTLA-4, while TGN1412 IgG4 does not.

[0049] Fig.11 The epitope recognized by AE2P is shown. Fig.11 A shows a scan of the membrane after incubation with AE2P IgG4. Fig.11 B shows the peptide array sequence of CD28 used for PepSpot. Dark grey highlights the dark spots on the membrane (arrays 24-25), while light grey highlights the light spots on the membrane (arrays 11-13). Fig.11 C and Fig.11 D is represented by a cartoon ( Fig.11 C) and surface representation ( Fig.11 D) shows the alignment of epitope mapping on CD28 crystal structures.

[0050] Fig.12 In vitro cell lysis of the WI-38 cell line was demonstrated using the bispecific T cell engager "L19-OKT3" alone or in combination with another bispecific T cell engager "7NP2-AE2P". The combination increased cell lysis of the WI-38 cell line, indicating a potent and specific synergy between the bispecific form of AE2P and the CD3-engaging bispecific antibody.

[0051] Fig.13 Some examples of molecular forms of AE2P that can be used for cancer immunotherapy and / or for treating chronic infectious diseases, autoimmune diseases, inflammatory disorders and / or preventing transplant rejection are presented.

[0052] Fig.14 Results of an in vitro superagonist activity assay performed on mouse T cells are shown. Data are presented as mean ± SD of technical triplicates. Fig.14 A shows the proliferation of mouse T cells after incubation with either AE2P IgG4, TGN1412 IgG4 (also referred to as TGN), or without antibody in the absence of anti-mouse CD3 antibody 2C11. This demonstrates the lack of proliferation of mouse T cells after 5 days of incubation with AE2P IgG4 alone. Fig.14 B shows the quantification of cytokines (IL-2 and IFN-γ) by ELISA after 3 days of incubation with either AE2P IgG4, TGN1412 IgG4 or without antibody in the absence of anti-mouse CD3 antibody 2C11. This confirms the lack of IL-2 and IFN-γ secretion by mouse T cells after 3 days of incubation with AE2P IgG4 alone. Fig.14 C shows the expression of mouse CD3 in the absence of anti-mouse CD3 antibody 2C11 after incubation with either AE2PIgG4 or TGN1412IgG4 for 3 days or without incubation with antibodies. + Expression of early activation markers (CD69) and late activation markers (CD25) of T cells. This confirmed the lack of upregulation of both CD69 and CD25 in mouse T cells after 3 days of incubation with AE2PIgG4 alone.

[0053] Fig.15 Results from an in vitro co-stimulation assay performed on mouse T cells are shown. Fig.15 A shows the proliferation of mouse T cells after incubation for 3 days with the anti-mouse CD3 antibody 2C11 alone or in combination with AE2P IgG4 and TGN1412 IgG4 (also referred to as TGN). This confirms that in the presence of antibody 2C11, AE2P IgG4 has a co-stimulatory effect on mouse T cell proliferation, while TGN1412 IgG4 does not. Fig.15 B shows the release of cytokines (IL-2 and IFN-γ) from mouse T cells after 3 days of incubation with 2C11 alone or in combination with AE2P IgG4 and TGN1412 IgG4. This confirms that AE2P IgG4 has a co-stimulatory effect on IL-2 secretion from mouse T cells when combined with 2C11 (left panel), while no changes in IFN-γ release were observed in the case of AE2P IgG4 or TGN1412 IgG4 (right panel).

[0054] Fig.16 Demonstrated anti-CD3 / anti-ED-B bispecific T cell engager (BiTE TM ) and the use of the anti-CD3 / anti-ED-B BiTE TM Results of in vitro co-stimulation and cell killing assays performed with the 247 molecules. Data are presented as mean ± SD (technical replicates n = 3). Fig.16 A shows the anti-CD3 / anti-ED-B BiTE TM Size exclusion chromatography results of anti-CD3 / anti-ED-B BiTE TM Eluted from the column as expected. Fig.16 B shows the anti-CD3 / anti-ED-B BiTETM SDS-PAGE analysis results of anti-CD3 / anti-ED-B BiTE TM The expected sizes were observed under non-reducing (NR) and reducing (R) conditions, respectively. Fig.16 C shows anti-CD3 / anti-ED-B BiTE as analyzed by flow cytometry TM Binding to effector human T cells (left) and target human cells WI-38 (right). Grey areas correspond to anti-CD3 / anti-ED-BBiTE TM The lines correspond to the fluorescence intensity of the isotype control. Fig.16 D shows the use of anti-CD3 / anti-ED-B BiTE alone TM "PUB4-L19", or the combination of "PUB4-L19" and TGN1412 IgG4 (also called TGN) or AE2P IgG4 for ED-B + Target cell lysis of the WI-38 target cell line. TM When "PUB4-L19" was used in combination with AE2P IgG4, a strong synergistic effect was observed, such as compared to using PUB4-L19 BiTE alone. TM As can be seen, an approximately two-fold increase in cell lysis was present. The stimulatory activity of AE2P IgG4 was only observed in the presence of the anti-CD3 / anti-ED-B BiTE TM (PUB4-L19) as a single agent was initially active only at concentrations (i.e., at 1 and 10 nM), unlike the superagonist TGN1412 (also called TGN) IgG4 antibody, which showed activity in the absence of CD3 co-stimulation (i.e., also at 0 and 0.1 nM). Fig.16 E) IFN-γ release ( Fig.16 F) and T cell proliferation ( Fig.16 Similar results were observed when G).

[0055] Fig.17 The anti-human CEA IgG / AE2P (scFv) 2 bispecific antibody (2+2; Fig.13 Biochemical characteristics of the form 1). Fig.17 A shows the result of size exclusion chromatography of the anti-human CEA IgG / AE2P (scFv) 2 bispecific antibody. The bispecific antibody was eluted from the S200i 10 / 300GL column as expected. Fig.17 B shows the results of SDS-PAGE analysis of the anti-human CEA IgG / AE2P (scFv) 2 bispecific antibody. The bispecific antibody has the expected size under non-reducing (NR) and reducing (R) conditions, respectively. Fig.17C shows the binding of anti-human CEA IgG / AE2P(scFv)2 bispecific antibody on primary mouse T cells as analyzed by flow cytometry. Fig.17 D shows the binding of anti-CEA IgG / AE2P(scFv)2 bispecific antibody on target cells expressing human CEA (C51.CEA).

[0056] Fig.18 The AE2P IgG / anti-human CEA (scFv)2 bispecific antibody (2+2; Fig.13 Biochemical characteristics of form 2). Fig.18 A shows the results of size exclusion chromatography of the AE2P IgG / anti-human CEA (scFv) 2 bispecific antibody. The bispecific antibody was eluted from the S200i 10 / 300GL column as expected. Fig.18 B shows the results of SDS-PAGE analysis of the AE2P IgG / anti-human CEA (scFv) 2 bispecific antibody. The bispecific antibody has the expected size under non-reducing (NR) and reducing (R) conditions, respectively. Fig.18 C shows the binding of AE2P IgG / anti-human CEA (scFv) 2 bispecific antibody on primary mouse T cells as analyzed by flow cytometry. Fig.18 D shows the binding of AE2P IgG / anti-human CEA (scFv) 2 bispecific antibody on target cells expressing human CEA (C51.CEA). DETAILED DESCRIPTION OF THE INVENTION

[0058] Various aspects and embodiments of the present invention will now be discussed with reference to the accompanying drawings. Other aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated herein by reference.

[0059] Antibody molecules

[0060] The present invention provides antibody molecules that bind to CD28. The antibody molecule binds to human CD28 and murine CD28, preferably to the extracellular domains of human CD28 and murine CD28. The extracellular domains of human CD28 and murine CD28 may comprise or consist of the sequences shown in SEQ ID NOs: 1 and 2, respectively. The antibody molecule is preferably capable of binding to CD28 expressed on the surface of cells (such as T cells). Methods for determining binding antigens (such as human or murine CD28) are known in the art and include, for example, ELISA and flow cytometry. The antibody molecule can bind to CD28 monovalently. The antibody molecule can bind to CD28 bivalently. The antibody molecule can be an agonist of CD28. The antibody molecule can be an antagonist of CD28.

[0061] The antibody molecule preferably specifically binds to CD28. The term "specificity" is applicable to situations where the antibody molecule is specific for a particular epitope (e.g., an epitope on CD28) that is carried by a variety of antigens, in which case the antibody molecule will be able to bind to various antigens carrying the epitope. For the purposes of the present invention, CTLA-4 carries the same "MYPPPY" binding motif present in the CD28 epitope to which the antibody binds. Therefore, the antibody is specific for a particular epitope that is present on both CD28 and CTLA-4.

[0062] The antibody molecule in the form of scFv preferably binds to human CD28 with high affinity. The antibody molecule may further bind to murine CD28. The antibody molecule in the form of scFv preferably has an EC of less than 35 nM for human CD28. 50 The EC of the antibody molecule to the cognate antigen (eg human CD28) 50 This can be determined by titration ELISA, for example as described in detail in the Examples.

[0063] The antibody molecule is preferably monoclonal. The antibody molecule may be human or humanized, but is more preferably a human antibody molecule.

[0064] The antibody molecule may be isolated in the sense of being free of contaminants (eg, antibodies capable of binding other polypeptides) and / or serum components.

[0065] The antibody molecule may be natural, or partially or completely produced by synthetic means. For example, the antibody molecule may be a recombinant antibody molecule.

[0066] The antibody molecule may be an immunoglobulin, or an antigen-binding fragment thereof. For example, the antibody molecule may be an IgG, IgA, IgE or IgM molecule, preferably an IgG molecule, such as IgG1, IgG2, IgG3 or IgG4 molecule, more preferably IgG1, IgG 2A Or an IgG4 molecule, more preferably an IgG4 molecule, or an antigen-binding fragment thereof, such as a single-chain antibody fragment (scFv), a diabody (Db), a single-chain diabody (scDb) or a small immune protein (SIP).

[0067] A single-chain antibody Fv molecule (scFv) is a molecule in which the VH domain and the VL domain are connected by a peptide linker that enables the two domains to bind to form an antigen binding site (Bird et al. (1988) Science, 242, 423-426; Huston et al. (1988) PNAS USA, 85, 5879-5883). Diabodies are multivalent or multispecific fragments constructed by gene fusion (WO2013 / 014149; WO94 / 13804; Holliger et al. (1993a), Proc. Natl. Acad. Sci. USA 90 6444-6448). Single-chain diabodies are molecules in which two sets of VH and VL domains are sequentially connected on the same polypeptide chain (Konterman and Muller, 1999). scFv or diabody molecules can be stabilized by incorporating disulfide bridges connecting the VH and VL domains (Reiter et al. (1996), Nature Biotech, 14, 1239-1245). Single-chain antibody Fv (scFv) can be contained within a small immune protein (SIP), such as described in (Li et al., (1997), Protein Engineering, 10: 731-736). The SIP can contain a CH4 domain (ε4) that is identical to the human IgE secretory isotype IgE S2. S2 -CH4; Batista et al., (1996), J. Exp. Med., 184: 2197-205) fused scFv molecules to form homodimeric mini-immunoglobulin antibody molecules.

[0068] In some embodiments, the antibody molecule may be an antigen binding fragment comprising an antigen binding site for CD28. The antigen binding site may be provided by one or more antibody variable domains (e.g., a so-called Fd antibody fragment consisting of a VH domain). Preferably, the antigen binding site comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0069] The antigen binding site of the antibody molecule (such as an immunoglobulin or an antigen-binding fragment thereof) of the present invention binds to CD28. The antigen binding site may comprise three CDRs, such as three light chain variable region (VL) CDRs or three heavy chain variable region (VH) CDRs, but preferably comprises six CDRs, i.e., three VL CDRs and three VH CDRs. The three VH domain CDRs of the antigen binding site may be located within the VH domain of the immunoglobulin, and the three VL domain CDRs may be located within the VL domain of the immunoglobulin. The antibody molecule may comprise one or two antigen binding sites for CD28. When the antibody molecule comprises two antigen binding sites, they are preferably identical. Thus, the antibody molecule may comprise one VH and one VL domain, but preferably comprises two VH and two VL domains, i.e., two VH / VL domain pairs, such as in naturally occurring immunoglobulin molecules, scFv, diabodies, and single-chain diabodies.

[0070] The antigen binding site of the antibody molecule preferably comprises three VL domain CDRs and / or three VH domain CDRs of the antibody AE2P. The VH and VL domain sequences of this antibody are shown in SEQ ID NOs: 9 and 10, respectively, and the CDR sequences of the AE2P antibody can be easily determined from these VH and VL domain sequences by a person skilled in the art using conventional techniques. For example, the CDR sequences can be determined according to Kabat et al., "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991). In a preferred embodiment, the antigen binding site of the antibody molecule comprises HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NOs: 3, 4 and 5, respectively, and LCDR1, LCDR2 and LCDR3 sequences shown in SEQ ID NOs: 6, 7 and 8, respectively.

[0071] In a further preferred embodiment, the antigen binding site may comprise the VH domain (SEQ ID NO: 9) and / or the VL domain (SEQ ID NO: 10) of antibody AE2P, but preferably comprises the VH domain and VL domain of antibody AE2P.

[0072] The antibody molecule may also comprise variants of CDR, VH domain, VL domain, heavy chain or light chain sequences as described herein. Suitable variants may be obtained by sequence alteration or mutation and screening methods. In a preferred embodiment, an antibody molecule comprising one or more such variant sequences retains one or more functional characteristics of the parent antibody molecule, such as binding specificity and / or binding affinity to human or murine CD28.

[0073] The antibody molecule may comprise a VH domain having at least 70%, more preferably at least 75%, 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 to the VH domain of antibody AE2P (SEQ ID NO:9).

[0074] The antibody molecule may comprise a VL domain having at least 70%, more preferably at least 75%, 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 to the VL domain of antibody AE2P (SEQ ID NO: 10).

[0075] The antibody molecule may comprise a heavy chain having at least 70%, more preferably at least 75%, 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 to the heavy chain of antibody AE2P in IgG1 form (SEQ ID NO: 13).

[0076] Antibody molecules can contain IgG 2A The heavy chain of antibody AE2P in the form of (SEQ ID NO: 17) has a heavy chain with at least 70%, more preferably at least 75%, 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.

[0077] The antibody molecule may comprise a heavy chain having at least 70%, more preferably at least 75%, 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 to the heavy chain of the IgG4 form of antibody AE2P (SEQ ID NO: 22).

[0078] The antibody molecule may comprise a light chain having at least 70%, more preferably at least 75%, 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 to the light chain of antibody AE2P (SEQ ID NO: 14).

[0079] Sequence identity is usually defined with reference to the algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences to maximize the number of matches and minimize the number of gaps. Default parameters are usually used, with a gap creation penalty of 12 and a gap extension penalty of 4. Use of GAP may be preferred, but other algorithms may be used, such as BLAST (which uses the method of Altschul et al. (1990) J. MoI. Biol. 215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981) J. MoI Biol. 147: 195-197), or the TBLASTN program of Altschul et al. (1990) (supra), generally using default parameters. In particular, the psi-BLAST algorithm (Nucl. Acids Res. (1997) 25 3389-3402) may be used.

[0080] Variants of the CDR, VH domain, VL domain, heavy chain or light chain sequences disclosed herein, comprising one or more (e.g., less than 20 changes, less than 15 changes, less than 10 changes or less than 5 changes, 4, 3, 2 or 1) amino acid changes (addition, deletion, substitution and / or insertion of amino acid residues), can also be used in the antibody molecules of the present invention. Suitable variants can be obtained by sequence changes or mutations and screening methods. Changes can occur in one or more framework regions and / or one or more CDRs. In particular, changes can be made in HCDR1, HCDR2 and / or HCDR3, or in one or more framework regions of the heavy chain or light chain of the antibody molecule.

[0081] As mentioned above, the antibody molecule may be a complete antibody or a fragment thereof, in particular an antigen-binding fragment thereof.

[0082] Preferably, the antibody molecule comprises a single-chain Fv (scFv), a small immune protein (SIP), a diabody, a single-chain diabody, a bispecific single-chain diabody (BiTE TM ) or (complete) IgG molecules (such as IgG1, IgG2A or IgG4 molecules), or consist of them.

[0083] When the antibody molecule is a scFv, the VH domain and VL domain of the antibody are preferably connected by a linker of 14 to 20 amino acids. For example, the VH and VL domains can be connected by an amino acid linker of 14, 15, 16, 17, 18, 19 or 20 amino acids in length. Suitable linker sequences are known in the art and include linker sequences as shown in SEQ ID NOs: 12 and 26.

[0084] In a preferred embodiment, the antibody molecule in the form of scFv of the present invention comprises or consists of the sequence shown in SEQ ID NO:11 or SEQ ID NO:25.

[0085] Diabodies are multimers of polypeptides, each polypeptide comprising a first domain containing an immunoglobulin light chain binding region and a second domain containing an immunoglobulin heavy chain binding region, the two domains being connected (e.g., by a peptide linker) but unable to bind to each other to form an antigen binding site; the antigen binding site is formed by the binding of the first domain of one polypeptide in the multimer to the second domain of another polypeptide in the multimer (WO94 / 13804; Holliger and Winter, 1997; Holliger et al., 1993).

[0086] In a diabody or single-chain diabody, the heavy chain variable region domain (VH) is linked to the light chain variable region domain (VL) on the same polypeptide chain. The VH and VL domains are connected by a peptide linker that is too short to allow pairing between the two domains, forcing them to pair with the complementary VH and VL domains on another chain.

[0087] When the antibody molecule is a diabody or a single-chain diabody, the VH and VL domains are preferably connected by a linker of 5 to 12 amino acids. For example, the VH and VL domains can be connected by an amino acid linker of 5, 6, 7, 8, 9, 10, 11 or 12 amino acids in length. Preferably, the amino acid linker is 5 amino acids in length. Suitable linker sequences are known in the art and include the linker sequence shown in SEQ ID NO: 21.

[0088] In a preferred embodiment, the antibody molecule in the form of a diabody of the present invention has the sequence shown in SEQ ID NO:19.

[0089] In a single-chain diabody, two groups of VH and VL domains are sequentially connected on the same polypeptide chain. For example, two groups of VH and VL domains can be assembled in the following single-chain sequence: (VH-VL)--(VH-VL), where brackets represent one group. The two groups of VH and VL domains are connected together as a single chain through a long or "flexible" peptide linker. This type of peptide linker sequence is long enough to allow the VH and VL domains of the first group to pair with the complementary VH and VL domains of the second group. Typically, the long or "flexible" linker is 15 to 20 amino acids long.

[0090] In a preferred embodiment, the antibody molecule of the present invention in the form of a single-chain diabody (scDb) has the sequence shown in SEQ ID NO:65.

[0091] When the antibody is a small immune protein (SIP) (e.g., as described in Li et al., (1997), Protein Engineering, 10: 731-736), the VL domain of the scFv antibody is preferably connected to the CH4 domain of human IgE (Batista et al., (1996), J. Exp. Med., 184: 2197-205) via a 2 to 20 amino acid linker, more preferably via a 2 to 10 amino acid linker. Suitable linker sequences are known in the art and include the linker sequence shown in SEQ ID NO: 16.

[0092] In another preferred embodiment, the antibody molecule in the form of SIP of the present invention has the sequence shown in SEQ ID NO:15.

[0093] Bispecific Binding Molecules

[0094] The antibody molecules of the present invention may constitute a part of a bispecific binding molecule (e.g., a bispecific antibody molecule). In some embodiments, the bispecific binding molecule comprises an antigen binding site that binds to a tumor-associated antigen, such as fibroblast activation protein (FAP), ED-A, ED-B or IIICS splicing isoforms of fibronectin, CAIX, splicing isoforms of tenascin-C (such as A1, A2, B, C or D isoforms of tenascin-C), mucin-16, PSMA or CEA. In a preferred embodiment, the bispecific binding molecule comprises an antigen binding site that binds to CEA. In a preferred embodiment of the replacement, the bispecific binding molecule comprises an antigen binding site that binds to FAP. In one embodiment, the second antigen binding site of the bispecific binding molecule may not bind to PSMA. In this embodiment, the second antigen binding site may be combined with FAP, ED-A, ED-B or IIICS splicing isoforms of fibronectin, CAIX, A1, A2, B, C or D isoforms of tenascin-C, mucin-16 or CEA.

[0095] Additionally, or alternatively, the bispecific binding molecule may comprise an antigen binding site that binds a second T cell antigen (eg, CD3).

[0096] The bispecific binding molecules according to the present invention are preferably bispecific antibodies and can be selected from IgG-added antibodies with additional antigen binding moieties (e.g. IgG-(scFv)2 or IgG-(scFv)) and small recombinant bispecific antibody formats (e.g. bispecific T cell engagers (BiTEs)). TM ) or scDb-scFv). Preferably, the bispecific antibody comprises specificity for a tumor-associated antigen (eg, CEA or FAP).

[0097] BiTE TM It is a single-chain diabody that contains two sets of different VH and VL domains, which form a bispecific single-chain diabody. Bispecific T cell engager (BiTE TM The sequences of the AE2P antibody and the anti-FAP antibody 7NP2 in the form of ) are shown in SEQ ID NO: 23. TM Examples of antibodies of the form are shown in Fig.13 in (see BiTE 1+1).

[0098] scDb-scFv is a single chain diabody that binds to a first target conjugated to a scFv, which binds to the scFv as a second target. The scFv can be conjugated to the VL domain of the single chain diabody (e.g., via an amino acid linker). In scDb-scFv, the AE2P antibody can be in the form of scDb or scFv. Thus, in one example, the AE2P antibody is in the form of scFv, and the scDb binds to a tumor-associated antigen. Alternatively, the AE2P antibody can be in the form of scDb, and the scFv binds to a tumor-associated antigen. Examples of antibodies in the form of scDb-scFv are shown in Fig.13 (see scDb-scFv(1+2)).

[0099] The IgG additional antibody comprising an additional antigen binding portion according to the present invention preferably comprises an IgG molecule and one or two scFvs. The scFv is preferably conjugated to the C-terminus of the heavy chain of the IgG molecule via an amino acid linker. When the IgG additional antibody comprises a single scFv conjugated to the C-terminus of one of the two heavy chains of the IgG molecule, the C-terminus of the other heavy chain is preferably free, i.e., unconjugated. This antibody format is also referred to as IgG-(scFv). IgG additional antibodies comprising scFv molecules conjugated to the C-termini of two heavy chains of the IgG molecule are also referred to as IgG-(scFv)2. These antibody formats are shown in Fig.13 (See IgG-(scFv)1+2 and IgG-(scFv)22+2 formats). In the IgG-added antibodies according to the present invention, the AE2P antibody can be in IgG or scFv format. Therefore, in one example, the AE2P antibody is in IgG format, and one or two heavy chains of the AE2P antibody in IgG format are conjugated to a scFv that binds to a tumor-associated antigen. Alternatively, the IgG-added antibody comprises an IgG that binds to a tumor-associated antigen, and one or two heavy chains of the IgG are conjugated to an AE2P antibody in scFv format. The heavy and light chain sequences of an anti-human CEA antibody and an AE2P antibody in IgG-(scFv)2 format, wherein the anti-human CEA antibody is in IgG format and the AE2P antibody is in scFv format ( Fig.13 1), as shown in SEQ ID NO: 61 and 62, respectively. The heavy chain and light chain sequences of AE2P antibody and anti-human CEA antibody in IgG-(scFv)2 format, wherein the AE2P antibody is in IgG format and the anti-human CEA antibody is in scFv format ( Fig.13 Form 2), as shown in SEQ ID NO:63 and 64, respectively.

[0100] Further examples of bispecific binding molecules can be found in Kontermann 2012 (p. 186 Figure 2 ), the contents of which are incorporated herein by reference.

[0101] Conjugate

[0102] The conjugate of the invention comprises an antibody molecule of the invention and a therapeutic agent or a diagnostic agent. The therapeutic agent may be a pro-inflammatory agent, a radioisotope, a photosensitizer, an enzyme or a hormone.

[0103] Proinflammatory cytokines that can be conjugated to the antibody molecules of the invention include interleukin-2 (IL2), interleukin-12 (IL12), interleukin-15 (IL15), interferons (such as IFNγ) and tumor necrosis factor (TNF), such as TNFα, and their mutants or variants.

[0104] The therapeutic agent can be conjugated to the N-terminus or C-terminus of the antibody molecule, or to both. When the therapeutic agent is conjugated to both the N-terminus and the C-terminus of the antibody molecule, the therapeutic agent can be the same or different, but preferably different. When the therapeutic agent is conjugated to the N-terminus of the antibody molecule, its C-terminus can be "free", i.e., not conjugated to another part. Similarly, when the therapeutic agent is coupled to the C-terminus of the antibody molecule, its N-terminus can be "free", i.e., not conjugated to another part.

[0105] The diagnostic agent conjugated to the antibody molecule of the invention may be a detectable label, such as a radioisotope, eg, a non-therapeutic radioisotope.

[0106] Radioisotopes that can be conjugated to the antibody molecules of the invention include isotopes such as 94m Tc, 99m Tc, 186 Re, 188 Re, 203 Pb, 67 Ga, 68 Ga, 47 Sc, 111 In, 97 Such as 62 Cu, 64 Cu, 86 Y. 88 Y. 90 Y. 121 Sn, 161 Tb, 153 Sm, 166 Ho, 105 Rh, 177 Lu, 123 I. 124 I. 125 I. 131 I. 18 F. 211 At and 225 Preferably, the positron emitter (such as18 F and 124 I) or gamma emitters (such as 99m Tc, 111 In and 123 I) are used for diagnostic applications (e.g. PET), while beta emitters (e.g. 131 I. 90 Y and 177 Lu) are preferably used for therapeutic applications. Alpha emitters (such as 211 At and 225 Ac) can also be used in therapy. In one example, the antibody molecule can be combined with 177 Lu, 131 I or 90 Y conjugated.

[0107] The antibody molecule can be conjugated to the therapeutic agent via a peptide bond or linker as described herein. Other means for conjugation include chemical conjugation, particularly cross-linking using bifunctional reagents (e.g., using DOUBLE-REAGENTS TM Cross-linking Reagents Selection Guide, Pierce).

[0108] Connectors

[0109] The antibody molecule (e.g., scFv or IgG) and the therapeutic or diagnostic agent or molecule can be directly linked to each other, for example, by any suitable chemical bond, but preferably by a peptide linker. The chemical bond can be, for example, a covalent bond or an ionic bond. Examples of covalent bonds include peptide bonds (amide bonds) and disulfide bonds.

[0110] When the therapeutic agent or diagnostic agent is connected to the antibody molecule via a peptide linker, the peptide linker can be a short (2-30, preferably 10-20) amino acid residue extension. Examples of suitable peptide linker sequences are known in the art. One or more different linkers can be used.

[0111] When the antibody molecule is linked to the therapeutic or diagnostic agent via a peptide bond or a peptide linker, the conjugate can be produced (secreted) as a single polypeptide chain (eg, a fusion protein).

[0112] Treatment

[0113] Therefore, the antibody molecules or conjugates of the present invention can be used as drugs.

[0114] In particular, the antibody molecule or conjugate may be used in a method of treatment of the human or animal body (which may include prophylactic treatment).

[0115] Also provided are methods of treating a disease or condition in a patient, wherein the method comprises administering to the patient a therapeutically effective amount of the antibody molecule or conjugate.

[0116] Further provided is the use of the antibody molecule or the conjugate in the preparation of a medicament for treating a disease or condition in a patient.

[0117] Treatment can be any treatment or therapy that achieves some desired therapeutic effect, such as inhibiting or slowing the progression of a disease or condition, and includes reducing the rate of progression, halting the rate of progression, ameliorating a disease or condition, curing or alleviating (whether partial or complete) a disease or condition, preventing, ameliorating, delaying, alleviating or arresting one or more symptoms and / or signs of a disease or condition, or prolonging the survival of an individual or patient beyond the expected survival of the individual or patient in the absence of treatment.

[0118] Treatment as a preventative measure (i.e., prophylaxis) is also included. For example, an individual who is susceptible to a disease or condition or is at risk of developing or recurring the disease or condition may be treated as described herein. Such treatment may prevent or delay the onset or recurrence of the disease or condition in the individual.

[0119] The method of treatment as described may include administering at least one other treatment to an individual in addition to the antibody molecule or conjugate. Therefore, the antibody molecule or conjugate may be administered to an individual alone, or may be administered in combination with one or more other treatments for the disease or condition. When an antibody molecule or conjugate is administered to an individual in combination with another treatment, the additional treatment may be administered to the individual simultaneously, sequentially or separately with the administration of the antibody molecule or conjugate. When the additional treatment is administered simultaneously with the antibody molecule or conjugate, the antibody molecule or conjugate and the additional treatment may be administered to the patient as a combined preparation. For example, the additional therapy may be a known therapy or therapeutic agent for a disease or condition to be treated. For example, the antibody molecule or conjugate of the present invention may be used in combination with a CD3 agonist (e.g., an antibody that binds human CD3) in a method of treatment as described herein.

[0120] Exemplary other therapeutic agents that can be used in combination with or administered in conjunction with the antibody molecules of the invention include, for example, chemotherapy (e.g., anti-cancer chemotherapy such as paclitaxel, docetaxel, vincristine, cisplatin, carboplatin or oxaliplatin), radiation therapy, checkpoint inhibitors targeting PD-1 (e.g., anti-PD-1 antibodies such as pembrolizumab, nivolumab or cemiplimab (see US 9,987,500)), checkpoint inhibitors targeting CTLA-4, LAG3 or TIM3, costimulatory agonist antibodies targeting, for example, GITR, OX40 or 4-1BB, and other costimulatory CD28 bispecific antibodies.

[0121] Diseases that can be treated using the antibody molecules or conjugates of the invention may be cancer, as well as other tumors and neoplastic conditions.

[0122] Exemplary cancers include any type of solid or non-solid cancer or malignant lymphoma, and particularly include liver cancer, lymphoma, leukemia (e.g., acute myeloid leukemia), sarcoma, skin cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, head and neck cancer, esophageal cancer, pancreatic cancer, kidney cancer, gastric cancer, and brain cancer. Cancer can be familial or sporadic. Cancer can be metastatic or non-metastatic.

[0123] Diseases that can be treated using the antibody molecules or conjugates of the present invention can be chronic infectious diseases. Exemplary chronic infectious diseases include chronic hepatitis B infection (HBV), human immunodeficiency virus (HIV) infection, and tuberculosis.

[0124] Autoimmune diseases that can be treated using the antibody molecules or conjugates of the invention include lupus erythematosus, rheumatoid arthritis and psoriatic arthritis.

[0125] An inflammatory or autoimmune disease that can be treated using the antibody molecule or conjugate of the invention is an inflammatory bowel disease (IBD), such as Crohn's disease or ulcerative colitis.

[0126] Diseases that can be treated using the antibody molecules or conjugates of the present invention can be transplant-related diseases. Transplant-related diseases that can be treated using the antibody molecules or conjugates of the present invention include transplant rejection, such as acute transplant rejection and chronic transplant rejection. Chronic transplant rejection includes graft-versus-host disease (GvHD).

[0127] Detection or diagnostic method

[0128] It is contemplated that the antibody molecules and conjugates are suitable for detecting CD28 in vivo and in vitro and are therefore useful in the imaging, detection and diagnosis of diseases characterized by or associated with the expression of CD28.

[0129] Therefore, the present invention also relates to the use of the antibody molecules or conjugates of the present invention for detecting CD28 on their cell surfaces in vitro or in vivo. The conjugate preferably comprises a detectable label to assist in detection. Alternatively, a secondary antibody or other detection reagent can be used to detect the binding of the antibody molecule to CD28. When the antibody molecule is conjugated to a radioisotope, scintigraphy can be used to detect the binding of the antibody molecule to CD28 in the patient.

[0130] Also provided is a method for detecting CD28 in vitro, the method comprising incubating the antibody molecule or conjugate with a sample obtained from an individual (e.g., a human patient), and detecting the binding of the antibody molecule or conjugate to the sample (e.g., T cells present in the sample), wherein the binding of the antibody molecule or conjugate to the sample indicates the presence of CD28. Methods for determining the binding of an antibody molecule or antigen to a sample are known in the art and include, for example, ELISA, flow cytometry, and immunostaining of tissue samples.

[0131] Further provided is the antibody molecule or conjugate for use in a method for detecting CD28 in vivo, the method comprising administering the antibody molecule or conjugate to an individual (eg, a human patient).

[0132] Pharmaceutical composition

[0133] Although the antibody molecule or conjugate can be administered alone, the antibody molecule and conjugate are usually administered in the form of a pharmaceutical composition. Therefore, another aspect of the invention relates to a pharmaceutical composition comprising at least one antibody molecule or conjugate of the invention and at least one other component, such as a pharmaceutically acceptable excipient. A method comprising formulating the antibody molecule or conjugate into a pharmaceutical composition is also provided.

[0134] In addition to the antibody molecule or conjugate, the pharmaceutical composition may also include a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. As used herein, the term "pharmaceutically acceptable" relates to compounds, materials, compositions and / or dosage forms that are suitable for contact with the tissue of a subject (e.g., human) and do not have excessive toxicity, irritation, allergic reaction or other problems or complications, and are consistent with a reasonable benefit / risk ratio within the scope of reasonable medical judgment. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the preparation. The exact nature of the carrier or other materials will depend on the route of administration, which can be by infusion, injection or any other suitable route, as discussed below.

[0135] For parenteral administration (e.g., subcutaneous or intravenous administration, e.g., by injection), the pharmaceutical composition comprising the antibody molecule or conjugate may be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has a suitable pH, isotonicity, and stability. A person skilled in the art will be able to prepare a suitable solution using, for example, an isotonic vehicle (e.g., sodium chloride injection, Ringer's injection, lactated Ringer's injection). Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be used as needed, including buffers such as phosphates, citrates, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride, phenol, butanol, or benzyl alcohol; alkyl parabens (e.g., methyl or propyl parabens), catechol, resorcinol, cyclohexanol, 3'-pentanol, and m-cresol); low molecular weight polypeptides; proteins (e.g., serum albumin, gelatin or immunoglobulin); hydrophilic polymers (such as polyvinyl pyrrolidone), amino acids (such as glycine, glutamine, asparagine, histidine, arginine or lysine); monosaccharides, disaccharides and other carbohydrates (including glucose, mannose or dextrin); chelating agents (such as EDTA); sugars or sugar alcohols (such as sucrose, mannitol, trehalose or sorbitol); salt-forming counterions (such as sodium ions); metal complexes (such as Zn-protein complexes) and / or non-ionic surfactants (such as TWEEN TM 、PLURONICS TM or polyethylene glycol (PEG).

[0136] In some embodiments, the antibody molecule or conjugate can be provided in a lyophilized form for reconstitution prior to administration. For example, the lyophilized antibody molecule or conjugate can be reconstituted in sterile water and mixed with saline before administration to an individual.

[0137] Administration can be in a "therapeutically effective amount", i.e., an amount sufficient to show benefit to an individual. The actual amount applied, the rate of administration, and the time course of administration will depend on the nature and severity of the disease or condition being treated, the specific individual being treated, the clinical condition of the individual, the cause of the condition, the delivery site of the composition, the type of antibody molecule or conjugate, the method of administration, the time schedule of administration, and other factors known to medical practitioners. The prescription of treatment, such as the determination of dosage, is within the responsibility of general practitioners and other doctors. Suitable doses of antibody molecules are well known in the art (Ledermann et al., 1991; Bagshawe et al., 1991). The specific doses specified herein, or the appropriate doses for the administered antibody molecules in the Physician's Desk Reference (2003) can be used. Suitable doses of conjugates are also known or can be determined. For example, the therapeutically effective amount or suitable dose of an antibody molecule or conjugate can be determined by comparing in vitro activity and in vivo activity in animal models (such as domestic dogs, pigs or sheep). Methods for extrapolating effective doses in domestic dogs, pigs and sheep and other test animals to humans are known. The exact dose will depend on many factors, including the size and location of the area to be treated, and the exact nature of the antibody molecule or conjugate.

[0138] Treatment may be repeated at daily, twice weekly, weekly or monthly intervals, at the physician's discretion. An individual's treatment regimen may depend on the pharmacokinetic and pharmacodynamic properties of the antibody molecule or conjugate, the route of administration, and the nature of the condition being treated.

[0139] Treatment can be cyclical, and the interval between two administrations can be about two weeks or longer, such as about three weeks or longer, about four weeks or longer, about monthly or longer, about five weeks or longer, or about six weeks or longer. For example, treatment can be every two to four weeks or every four to eight weeks (once). Suitable formulations and routes of administration have been described above.

[0140] The pharmaceutical composition may be administered alone or in combination with other therapies, either simultaneously or sequentially, depending on the condition to be treated.

[0141] Reagent test kit

[0142] Another aspect of the invention provides a therapeutic kit for use in the treatment of a disease or condition, comprising an antibody molecule or conjugate as described herein. The components of the kit are preferably sterile and placed in sealed vials or other containers.

[0143] The kit may also include instructions for use of the components in the methods described herein.The components of the kit may be contained or packaged in a container, such as a bag, box, can, tin or blister pack.

[0144] Nucleic acid, vector, host cell and production method

[0145] An isolated nucleic acid molecule encoding an antibody molecule or conjugate of the invention is provided. The nucleic acid molecule may comprise DNA and / or RNA and may be partially or fully synthetic.

[0146] The isolated nucleic acid molecules can be used to express the antibody molecules or conjugates of the present invention. The nucleic acid will usually be provided in the form of an expression vector. Another aspect of the present invention therefore provides an expression vector comprising a nucleic acid as described above. A suitable vector can be selected or constructed, comprising appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other appropriate sequences. Preferably, the vector comprises appropriate regulatory sequences that drive the expression of the nucleic acid in the host cell. The vector can be a plasmid, a virus (e.g., a phage) or a phagemid, as appropriate in the context.

[0147] Nucleic acid molecules or expression vectors as described herein can be introduced into host cells. The technology of introducing nucleic acids or vectors into host cells is well established in the art and any suitable technique can be used. A range of host cells suitable for producing recombinant antibody molecules and conjugates are known in the art, including bacteria, yeast, insect or mammalian host cells. Preferred host cells are mammalian cells, such as CHO cells, NS0 or HEK cells, for example HEK293 cells.

[0148] Another aspect of the present invention provides a method for producing an antibody molecule or a conjugate, the method comprising expressing a nucleic acid encoding an antibody molecule or a conjugate in a host cell, and optionally separating and / or purifying the antibody molecule or the conjugate produced thereby. Methods for culturing host cells are well known in the art. The method may further comprise separating and / or purifying the antibody molecule or the conjugate. Purification techniques for recombinant antibody molecules or conjugates are well known in the art, and include, for example, HPLC, FPLC or affinity chromatography (e.g., using protein A or protein L). In some embodiments, affinity tags on the antibody molecule may be used for purification. The method may also comprise formulating the antibody molecule or the conjugate into a pharmaceutical composition, optionally formulated with a pharmaceutically acceptable excipient or other substances as described herein.

[0149] The features disclosed in the foregoing description, the attached claims or the drawings, expressed in their specific form, or expressed in terms of means for performing the disclosed functions, methods or processes for obtaining the disclosed results (as the case may be), may be used alone or in any combination to implement the invention in a variety of forms.

[0150] Although the present invention is described in conjunction with the above exemplary embodiments, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Therefore, the exemplary embodiments of the present invention set forth above should be considered illustrative, rather than restrictive. Various changes may be made to the embodiments without departing from the spirit and scope of the present invention.

[0151] To avoid any doubt, any theoretical explanations provided herein are intended to improve the reader's understanding and the inventors do not wish to be bound by such theoretical explanations.

[0152] Any section headings used herein are for the purpose of organizing content only and are not to be construed as limiting the subject matter described.

[0153] Throughout the specification (including the following claims), unless the context requires otherwise, the words "comprise" and "include" and variations such as "comprises", "comprising" and "including", will be understood to imply the inclusion of a stated integer, step or group of integers or steps but not the exclusion of any other integer, step or any other group of integers or steps.

[0154] It must be noted that the singular forms "a", "an", "the" used in the specification and the appended claims include plural referents unless the context clearly indicates otherwise. Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes a range from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by using the prefix "about", it is understood that the particular value forms another embodiment. The term "about" in reference to a value is optional and means, for example, + / - 10%. Example

[0155] Example 1 - Cloning of human CD28 and isolation of AE2P by phage display.

[0156] 1.1 Expression process

[0157] Transient gene expression (TGE) in CHO-S cells was used to express a recombinant fragment of human CD28-ECD (extracellular domain) containing a C-terminal Avi tag, a His6 tag, and an Fc tag ("CD28-ECD" is also interchangeably referred to as "CD28-Fc" and "CD28-Fc fusion protein" herein). For a yield of 1 mL, 4×106 suspended CHO-S cells were centrifuged and resuspended in 1 mL of an appropriate culture medium. 0.9 μg of plasmid DNA per million cells was then added to the cells, followed by 2.5 μg of polyethyleneimine (PEI; 1 mg / mL aqueous solution at pH 7.0) and gently mixed. The transfected culture was placed in a shaking incubator and incubated at 31°C for 6 days. The protein fragment was purified from the cell culture medium using protein A affinity chromatography, then dialyzed into PBS buffer and stored at -80°C.

[0158] The amino acid sequence of the CD28-Fc fusion protein is shown in SEQ ID NO:66.

[0159] 1.2 Antigen characterization

[0160] By SDS-PAGE and Human CD28-ECD fusion protein was analyzed by size exclusion chromatography on FPLC using a Superdex 200 Increase (S200i) 10 / 300GL column. Binding of anti-CD28 antibodies to the recombinant antigen was verified by ELISA.

[0161] 1.3 Antigen biotinylation

[0162] Use BirA (E. coli biotin ligase) to carry out site-specific biotinylation to the purified human CD28-ECD fusion protein. First, the protein is dialyzed in BirA buffer (100mM Tris, pH 7.5, 200mM NaCl, 5mM MgCl). By adding 1mg protein, 28μL 40mM biotin, 43μg BirA, 70μL 0.5M ATP and protease inhibitors, the biotinylation reaction of 24 hours is carried out. The next day, the protein is purified by size exclusion chromatography and dialyzed back into PBS buffer.

[0163] 1.4 Phage display selection

[0164] Bio-panning was performed using biotinylated human CD28-ECD fusion protein using streptavidin magnetic beads. Briefly, biotinylated human CD28-ECD fusion protein (final concentration of 120 pmol) was incubated with 800 μL pre-blocked phage display library for 30 minutes. After washing several times with PBS buffer, the bound phage was eluted by changing the pH using triethylamine. The separated phage was then amplified in Escherichia coli strain TG-1 and precipitated from the supernatant using polyethylene glycol.

[0165] After two rounds of bio-panning, clones were screened by ELISA. Avidin-coated ELISA plates were incubated with biotinylated human CD28-ECD fusion proteins. To exclude phages bound to Fc, biotinylated human IgG was also used in parallel to coat avidin-coated ELISA plates. The supernatant of the monoclonal clones of the selected induction in the Escherichia coli TG-1 culture expressing the scFv antibody fragment was added to two ELISA plates, and anti-c-myc antibody 9E10 was then used to detect the bound scFv using anti-mouse IgG-horseradish peroxidase (HRP) conjugates.

[0166] Example 2 - Biochemical Characterization

[0167] 2.1 In vitro characterization of AE2P antibody in scFv format

[0168] AE2P scFv was produced in E. coli strain TG-1. TG-1 cultures were grown in 2xTY / 100 μg / mL ampicillin at 37°C. At OD600 = 0.5, 1 mM isopropyl-β-D-thiogalactoside (IPTG) was added to induce expression of the scFv; the culture was incubated overnight at 30°C with shaking at 175 rpm in a bacterial incubator. The culture was then centrifuged, purified from the supernatant of the cell culture medium by protein A affinity chromatography, then dialyzed against PBS buffer, and stored in PBS at -80°C. The scFv was then used AE2P scFv was characterized and purified by size exclusion chromatography on an S75i 10 / 300GL column on FPLC ( Figure 1 A). SDS-PAGE analysis was also performed using 4-12% Bis-Tris gels under reducing and non-reducing conditions ( Figure 1 B). Titration ELISA was performed by coating biotinylated CD28-ECD fusion protein or biotinylated nonspecific protein on streptavidin wells. After blocking with 2% milk-PBS, various concentrations of AE2P scFv were added and the signal was detected using mouse anti-myc tag followed by anti-mouse HRP. S-shaped curves were plotted at different concentrations ( Figure 1C). These results indicate that AE2P scFv has the expected molecular weight under both reducing and non-reducing conditions and eluted from the SEC column at 11.5 mL, with the single peak observed by SEC indicating excellent purity. Titration ELISA showed EC 50 The p-value of AE2P scFv was about 31 nM, which confirmed the binding of AE2P scFv to CD28.

[0169] 2.2 In vitro characterization of AE2P antibody in IgG4 format

[0170] The AE2P antibody in IgG4 form was produced by the same method as described above. Specifically, AE2P IgG4 was cloned, expressed using transient gene expression (TGE) in CHO-S cells, purified by protein A affinity chromatography, dialyzed, and stored in PBS. AE2P IgG4 was characterized by size exclusion chromatography on a S200i 10 / 300GL column on FPLC ( Figure 2 A). SDS-PAGE analysis was also performed using 4-12% Bis-Tris gels under reducing and non-reducing conditions ( Figure 2 B). Flow cytometry analysis of primary human T cells was performed by staining with 50 nM AE2P IgG4, TGN1412 IgG4 (as defined in SEQ ID NO: 27 and 28; US Patent 8709414) or isotype IgG4. Cells were first blocked in FACS buffer (FBS, 2% FBS and 2 mM EDTA) for 30 minutes. After incubation with primary antibody for 1 hour, cells were washed twice with FACS buffer, and secondary anti-human PE antibody was added to detect fluorescence shift in the case of binding ( Figure 2 C). Use near infrared (NIR) Zombie to distinguish live cells from dead cells. Flow cytometry analysis of various cell lines was performed in a similar manner as described above by staining with 50 nM AE2P IgG4 or isotype IgG4. Secondary antibodies anti-human PE or anti-human Alexa 488 antibodies were added to detect the fluorescence shift in the case of binding ( Figure 3 ). These results indicate that AE2P IgG4 has the expected molecular weight under both reducing and non-reducing conditions and elutes from the SEC column at 11.9 mL, with the single peak observed by SEC indicating excellent purity. Figure 2 Flow cytometric analysis in C shows that both AE2P IgG4 and TGN1412 IgG4 bind to primary human T cells expressing CD28. Figure 3 Flow cytometric analysis showed that AE2P IgG4 specifically bound to primary human T cells but not to other cell lines that did not express CD28.

[0171] Example 3 - Co-stimulation assay in combination with anti-CD3 monoclonal antibody "OKT3

[0172] 3.1 Cell proliferation in combination with OKT3

[0173] The co-stimulatory effects of AE2P and TGN1412 antibodies were tested by comparing the extent of cell proliferation induced by each antibody in the presence of CD3 stimulation. TM ) is an activating monoclonal antibody against the CD3 receptor. OKT3IgG4 was coated on a 6-well plate (1 μg per 1 mL, 2 mL in total) for 2 hours at 37°C in a cell culture incubator. Purified human PBMCs from healthy donors (500,000 per mL in 3 mL) were added to 6-well plates (triplicates for each experimental condition). AE2P IgG4, TGN1412 IgG4, or isotype IgG4 at a concentration of 5 μg per 1 mL were added to each experimental condition in solution form. Human PBMCs without added antibodies were also included as controls. The culture plates were incubated at 37°C in a cell culture incubator for 3 days. For cell proliferation, 200 μL from each triplicate was added to a 96-well plate, 20 μL of CellTiter96Aqueous One Solution cell proliferation assay was added, and incubated at 37°C in a cell culture incubator for 2 hours. The absorbance at 490 nM was measured, and the percentage increase in cell proliferation (normalized to control conditions) was plotted ( Figure 4 ). These results show the co-stimulatory effect on human PBMCs produced by the combination of anti-CD28 antibodies (AE2P IgG4 and TGN1412 IgG4) and anti-CD3 antibodies (OKT3). In particular, it was shown that the increase in cell proliferation in the presence of AE2P IgG4 was greater than the increase in cell proliferation achieved with TGN1412 IgG4, indicating that AE2P IgG4 produced a stronger co-stimulatory effect.

[0174] 3.2 Cytokine release in combination with OKT3

[0175] The co-stimulatory effects of AE2P and TGN1412 antibodies were further tested by comparing the extent of cytokine release induced by each antibody in the presence of CD3 stimulation. Specifically, cells from the above assay (Section 3.1) were centrifuged at 500 g for 10 min, and the supernatant was transferred and stored at -20°C. IL-2 ( Figure 5 A) IFN-γ ( Figure 5 B) TNFα ( Figure 5C) cytokine release. These results show that when anti-CD28 antibodies (AE2P IgG4 and TGN1412 IgG4) are used in combination with anti-CD3 antibodies (OKT3), IL-2, IFN-γ, and TNF-α secretion from human PBMCs is increased. For these three cytokines, the increase in secretion in the presence of AE2P IgG4 is greater than the increase in secretion in the presence of TGN1412 IgG4, indicating that AE2P IgG4 provides a stronger costimulatory effect.

[0176] Example 4 - Superagonism Assay of AE2P

[0177] 4.1 Cell proliferation

[0178] The super-agonist activity of AE2P and TGN1412 antibodies was tested by comparing the degree of cell proliferation induced by each antibody in the absence of CD3 stimulation. Specifically, AE2P antibody in IgG4 form, TGN1412 antibody in IgG4 form, or isotype IgG4 was coated on a 6-well plate (3 μg per 1 mL in 2 mL) for 2 hours in a cell culture incubator at 37°C. Purified human PBMCs from healthy donors (500,000 per mL in 3 mL) were added to 6-well plates (triplicate for each experimental condition). Human PBMCs without added antibodies were also included as controls. The culture plates were incubated at 37°C in a cell culture incubator for 3 days. On day 3 ( Figure 6 A) and 5 days ( Figure 6 B) After that, cell proliferation assay was performed as described above (Section 3.1). These results indicate that, unlike TGN1412 IgG4, AE2P IgG4 does not induce proliferation of human PBMCs in the absence of CD3 stimulation, demonstrating that AE2P does not have superagonist activity.

[0179] 4.2 Cytokine release

[0180] The superagonist activity of AE2P and TGN1412 antibodies was further tested by comparing the extent of cytokine release induced by each antibody in the absence of CD3 stimulation. Specifically, cells from the above experiment (Section 4.1) were centrifuged at 500 g for 10 minutes, and the supernatant was transferred and stored at -20°C. IL-2 ( Figure 7 A) IFNγ( Figure 7 B) and TNF-α( Figure 7 C) Cytokine release. These results indicate that, unlike TGN1412 IgG4, AE2P IgG4 does not induce secretion of these three cytokines (IL-2, IFNγ, and TNF-α) by human PBMCs, confirming that this antibody does not have superagonist activity.

[0181] 4.3 Expression of activation markers

[0182] The super-agonist activity of AE2P and TGN1412 antibodies was further tested by comparing the degree of upregulation of activation markers induced by each antibody in the absence of CD3 stimulation. Specifically, cells from the above experiment (Section 4.1) were centrifuged at 500g for 10 minutes. The expression of activation markers (CD69 and CD25) after 3 days was evaluated by flow cytometry using the following antibody panel (CD4-FITC, CD8-BV421, CD69-PE, and CD25-Alexa 647). Zombie NIR was added to distinguish between live and dead cells. The data were plotted as the percentage of CD69 expression in CD4 positive cells ( Figure 8 A) The percentage of CD69 expression in CD8 positive cells ( Figure 8 B) The percentage of CD25 expression in CD4 positive cells ( Figure 8 C) and the percentage of CD25 expression in CD8 positive cells ( Figure 8 D). These results indicate that, unlike TGN1412 IgG4, AE2P IgG4 does not induce upregulation of activation markers CD69 and CD25 in CD4-positive T cells and CD8-positive T cells, confirming that this antibody does not have superagonist activity.

[0183] Example 5 - Cross-reactivity of AE2P with murine CD28 ECD

[0184] 5.1AE2P (IgG4) binding to human and mouse CD28 ECD

[0185] To test whether the AE2P antibody also binds to murine CD28, a titration ELISA was performed by coating CD28-ECD protein containing a His6 tag or a nonspecific protein on NiNTA-coated plates. After blocking with 2% milk-PBS, various concentrations of FITC-conjugated AE2P IgG4 were added, and the signal was detected using rabbit anti-FITC antibody followed by goat anti-rabbit HRP. Fig. 9 A) and murine CD28 ( Fig. 9 B) The binding of AE2P IgG4 to human and murine CD28 Fc was plotted as a sigmoidal curve. These results indicate that AE2P IgG4 is able to bind to both human and murine CD28 Fc.

[0186] 5.2 Binding of TGN1412 (IgG4) to human and mouse CD28 ECD

[0187] To test whether the TGN1412 antibody also binds to murine CD28, a titration ELISA was performed by coating CD28-ECD protein or nonspecific protein containing a His6 tag on NiNTA-coated plates. After blocking with 2% milk-PBS, different concentrations of FITC-conjugated TGN1412 IgG4 were added, and the signal was detected using rabbit anti-FITC antibody followed by goat anti-rabbit HRP. Fig. 9 C) and murine CD28 ( Fig. 9 D) The binding of the IgG4 protein to human CD28 Fc was plotted as a sigmoidal curve. These results indicate that TGN1412 IgG4 binds to human CD28 Fc but not to murine CD28 Fc.

[0188] 5.3 Binding to primary mouse T cells

[0189] To further test whether AE2P and TGN1412 antibodies also bind to murine CD28 expressed on cells, flow cytometry analysis of primary mouse T cells was performed by staining with 50 nM AE2P IgG4, TGN1412 IgG4, or isotype IgG4. Cells were first blocked in FACS buffer (PBS, 2% FBS and 2 mM EDTA) for 30 minutes. After incubation with primary antibodies for 1 hour, cells were washed twice with FACS buffer, and secondary anti-human PE antibodies were added to detect fluorescence shifts in the presence of binding ( Fig. 9 E). Zombie NIR is used to distinguish live from dead cells. The fluorescence shift caused by AE2P binding to primary mouse T cells indicates binding to murine CD28 on the surface of these cells, while the lack of fluorescence shift observed in the case of TGN1412 indicates the lack of TGN1412 binding to primary mouse T cells.

[0190] 5.4 Binding to human CTLA-4

[0191] To test whether AE2P and TGN1412 antibodies also bind to CTLA-4, a titration ELISA was performed by coating human CTLA-4 protein (SinoBiological, Cat: 11159-H08H) containing a His6 tag on NiNTA-coated plates. After blocking with 2% milk-PBS, various concentrations of FITC-conjugated AE2P IgG4 or TGN1412 IgG4 were added, and the signal was detected using rabbit anti-FITC antibody followed by goat anti-rabbit HRP. Data are plotted for binding to human CTLA-4 at different concentrations ( Fig.10 ). The results showed that AE2P IgG4 bound to human CTLA-4, while TGN1412 IgG4 did not.

[0192] 5.5 In vitro superagonist activity assay on mouse T cells

[0193] To test whether AE2P and TGN1412 antibodies have super-agonist activity in a mouse environment, the extent of mouse T cell proliferation induced by each antibody in the absence of CD3 stimulation was compared. Specifically, AE2P antibody in IgG4 form and TGN1412 antibody in IgG4 form (used as an isotype negative control here because it has no cross-reaction with murine CD28) were coated on 6-well plates (10 μg per well). Primary mouse T cells (500,000 per 1 mL in 3 mL) were added to 6-well plates (triplicate for each experimental condition). Primary mouse T cells without added antibodies were also included as controls. The culture plates were incubated in a cell culture incubator at 37°C for 3 or 5 days. On day 5 ( Fig.14 A), cell proliferation assays were performed as described above (Section 3.1). These results indicate that AE2P IgG4 does not induce proliferation of mouse T cells in the absence of CD3 stimulation, demonstrating that AE2P does not have superagonist activity in a mouse setting.

[0194] Fig.14 B shows the quantification of cytokines (IL-2 and IFN-γ) by ELISA after 3 days, which was performed as described in Section 4.2 except for the above differences. These results show that AE2P IgG4 does not induce IL-2 or IFN-γ secretion by mouse T cells, confirming that this antibody does not have superagonist activity in the mouse environment.

[0195] Fig.14 C shows the evaluation of activation markers CD69 and CD25 by flow cytometry after 3 days, which was performed as described in Section 4.3 except for the above differences. These results show that AE2P IgG4 does not induce upregulation of activation markers CD69 and CD25 in mouse T cells, confirming that this antibody does not have superagonist activity in the mouse setting.

[0196] In summary, no mouse T cell activation was observed with the AE2PIgG4 antibody in cell proliferation assays, cytokine quantification assays, or activation marker assays. Taken together, these results confirm that this antibody does not have superagonist activity in a mouse setting.

[0197] 5.6 In vitro co-stimulation assay of mouse T cells in combination with anti-murine CD3 monoclonal antibody 2C11

[0198] The co-stimulatory effects of AE2P and TGN1412 antibodies were tested by comparing the extent of mouse T cell proliferation induced by each antibody in the presence of CD3 stimulation. Specifically, 2C11, an activating monoclonal antibody for murine CD3 receptor (clone: ​​145-2C11, Biolegend, 100340), was coated on a 6-well plate (5 μg per 1 mL in 2 mL) for 2 hours in a cell culture incubator at 37 ° C. Primary mouse T cells (500,000 per 1 mL in 3 mL) were added to 6-well plates (triplicate for each experimental condition). AE2P IgG4 and TGN1412 IgG4 (used as isotype negative controls here) at a concentration of 5 μg per 1 mL (a total of 15 μg) were added to each experimental condition. Primary mouse T cells without added antibodies were also included as controls. The culture plates were incubated at 37 ° C in a cell culture incubator for 3 days. For cell proliferation, 200 μL from each triplicate was added to a 96-well plate, 20 μL of CellTiter 96 Aqueous One Solution Cell Proliferation Assay was added, and incubated in a cell culture incubator at 37°C for 2 hours. The absorbance at 490 nM was measured and the percentage increase in cell proliferation (normalized to control conditions) was plotted ( Fig.15 A). These results indicate that only the AE2P IgG4 antibody, but not the TGN1411 IgG4 antibody, exerts a co-stimulatory effect on the proliferation of mouse T cells in combination with the anti-CD3 antibody (2C11).

[0199] The co-stimulatory effects of the AE2P and TGN1412 antibodies were further tested in a mouse setting by comparing the extent of cytokine release induced by each antibody in mouse T cells in the presence of CD3 stimulation, which was performed as described above (Section 3.2) except for the above differences. The results showed that AE2P IgG4, but not TGN1412 IgG4, induced a significant increase in IL-2 production in mouse T cells when combined with the anti-murine CD3 antibody 2C11 ( Fig.15 B, left panel). No changes in IFN-γ release were observed for AE2P IgG4 and TGN1412 IgG4 ( Fig.15 B, right).

[0200] Example 6-Epitope mapping of AE2P

[0201] Epitope mapping was performed using synthetic peptides prepared by SPOT-synthetic scanning of the CD28 ECD sequence ( Fig.11 B). The experiment was performed by adding 2 μg / mL of AE2P IgG4. The membrane was scanned to show the important binding areas (sites) of AE2P ( Fig.11A). The potential binding residues are superimposed on the published CD28 crystal structure (PDB: 1YJD) (Evans et al., Nat Immunol, 2005, 6(3): 271-9), and a cartoon schematic is shown ( Fig.11 C) and surface schematic ( Fig.11 D) These results demonstrate the CD28 epitope recognized by the AE2P antibody and the location of this epitope on the CD28 crystal structure.

[0202] Example 7 - In vitro killing assay using AE2P bispecific antibody

[0203] To test the efficacy of the bispecific AE2P antibody, AE2P was expressed as a bispecific T cell engager (BiTE TM ) antibody format was cloned together with the antigen binding site of the tumor-targeting anti-FAP antibody "7NP2" (WO2022 / 223824), and with another BiTE containing the antigen binding site of the tumor-targeting anti-EDB antibody "L19" (US Patent No. 8,097,254) and the anti-CD3 antibody OKT3 TM Molecular combinations were tested.

[0204] The WI-38 cell line expressing EDB, FAP, and GFP was used in an in vitro specific cell lysis experiment using primary human T cells. 15,000 WI-38 cells were coated on a 96-well plate and 75,000 primary human T cells were added. In triplicate, the "L19-OKT3" BiTE was added at a concentration of 0.1 nM alone. TM , or by mixing it with 10 nM of "7NP2-AE2P" BiTE TM After 48 hours, all T cells and target cells were collected, washed with PBS, and then incubated with Zombie NIR for 30 minutes to distinguish live cells from dead cells. Cells were washed once with FACS buffer and collected for flow cytometry analysis. WI-38 cells were gated based on GFP and the percentage of specific cell lysis was calculated ( Fig.12 ). These results suggest that, compared with the “L19-OKT3” BiTE alone TM Compared with the "7NP2-AE2P" BiTE TM BiTE binding to CD3 TM The (L19-OKT3) combination resulted in increased specific cell lysis of the WI-38 cell line. This indicates a strong and specific synergy between the bispecific form of AE2P and the CD3-binding bispecific antibody.

[0205] Example 8 - Different forms of AE2P

[0206] AE2P is expected to have broad applications in cancer immunotherapy (i.e., as an enhancer in immunotherapy due to CD28 agonism and as an immune checkpoint inhibitor due to CTLA-4 binding), as well as in the treatment of chronic infectious diseases due to its ability to stimulate the immune system. Fig.13 Several potential monospecific, bispecific, or multispecific formats of AE2P are demonstrated, with or without conjugation to binding moieties specific for tumor targets.

[0207] Example 9 - BiTE TM Cloning, characterization and activity of anti-CD3 / anti-EDB

[0208] 9.1 Cloning and Characterization of Anti-CD3 / Anti-EDB Bispecific Antibodies

[0209] Anti-CD3 / anti-EDB BiTE TM (Clone PUB4 for anti-CD3 (as described by Liu Y, et al. (2022)), clone L19 for anti-EDB) were cloned into the mammalian expression vector pcDNA3.1+ with an N-terminal ×6His tag. Anti-CD3 / anti-EDB BiTEs were purified by protein A affinity chromatography TM . By SDS-PAGE and Purified BiTEs were analyzed by size exclusion chromatography using a Superdex 75 Increase (S75i) column on FPLC. TM Validation of the anti-CD3 / anti-EDB BiTE by flow cytometry on target cells (WI-38) and effector cells (purified human T cells) TM The combination of .

[0210] Anti-CD3 / anti-EDB BiTE TM The sequence of the molecule is shown in SEQ ID NO:60. Fig.16 A shows anti-CD3 / anti-ED-BBiTE TM As expected, anti-CD3 / anti-EDB BiTE TM Elute from the column. Fig.16 B shows the anti-CD3 / anti-ED-B BiTE TM SDS-PAGE analysis results of anti-CD3 / anti-ED-B BiTE TM The expected sizes were observed under non-reducing (NR) and reducing (R) conditions, respectively. Fig.16 C shows anti-CD3 / anti-ED-BBiTE as analyzed by flow cytometry TM Binding to effector human T cells (left panel) and target human cell WI-38 (right panel). Grey area corresponds to anti-CD3 / anti-EDB BiTE TMThe lines correspond to the fluorescence intensity from the isotype control.

[0211] 9.2 In vitro co-stimulation and cell killing assays in combination with anti-CD3 / anti-EDB bispecific antibodies

[0212] By comparing the presence of anti-CD3 / anti-EDB BiTE TM The co-stimulatory effects of AE2P and TGN1412 antibodies on human PBMCs were tested by measuring the extent of T cell proliferation and target cell killing induced by each antibody in the presence of CD3 stimulation.

[0213] Specifically, WI-38 cells expressing the extracellular domain B (EDB) of fibronectin and eGFP were coated on 96-well plates (20,000 cells / well). Fresh frozen human PBMCs (effector cells) were added to obtain a 5:1 effector cell to target cell ratio. Different concentrations of anti-CD3 / anti-EDB BiTE were added in 10-fold serial dilutions (10, 1, and 0.1 nM). TM . Also included is no added BiTE TM The conditions were used as negative controls. AE2P IgG4 and TGN1412 IgG4 at a fixed concentration of 50 nM were added to each condition. After 4 days, the cells were stained with Accutase TM Cells were detached with cell detachment solution. The supernatant was collected to measure IFN-γ release by ELISA. The remaining cell clumps and debris were washed with PBS, then live / dead stained with Zombie Violet and incubated at 4°C in the dark for 30 minutes. The cells were washed once and stained with FACS containing anti-human CD3 APC and anti-human CD25 Alexa Fluor TM 647 antibody master mix. Target cells were distinguished from effector cells by their expression of eGFP, and the percentage of dead cells was calculated by gating on dead cells. Live CD3 + The absolute number of T cells was used to assess proliferation and CD3 + Percentage of CD25 expression on T cells.

[0214] The results showed that anti-CD3 / anti-EDB BiTE alone TM Moderate target cell lysis was induced at concentrations of 1 nM and 10 nM (33% and 47% dead cells, respectively), but no effect was observed at concentrations of 0 nM and 0.1 nM ( Fig.16 D) AE2P IgG4 and anti-CD3 / anti-EDB BiTE relative to negative control TMThe combination resulted in an approximately doubling of the percentage of target cell lysis. However, unlike TGN1412IgG4, TGN1412 IgG4 was significantly more lysable in all anti-CD3 / anti-EDB BiTEs relative to the negative control. TM However, for AE2P IgG4, this synergistic effect was only observed in the anti-CD3 / anti-EDB BiTE TM Anti-CD3 / anti-EDB BiTEs active when added as single agents TM This was observed only at 1 nM and 10 nM concentrations. This suggests that, unlike TGN1412 IgG4, AE2P IgG4 is active only in the presence of CD3 stimulation. Fig.16 E) IFN-γ release ( Fig.16 F) and T cell proliferation ( Fig.16 Similar results were obtained in G). Data were statistically analyzed by one-way ANOVA followed by Tukey's multiple comparison test (B&C) or by two-way ANOVA followed by Dunnett's multiple comparison test (EH). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0215] Example 10 - Anti-human CEA IgG / anti-CD28 (scFv) 2 bispecific antibody ( Fig.13 The clones and expressions of form 1) Levy

[0216] Cloned IgG-(scFv)2(2+2) format ( Fig.13 The gene of the anti-human CEA IgG / AE2P (scFv) 2 bispecific antibody of form 1) was expressed using transient gene expression (TGE) in CHO-S cells, purified by protein A affinity chromatography, dialyzed and stored in PBS buffer. The bispecific antibodies were characterized by size exclusion chromatography using a S200i 10 / 300GL column on FPLC. SDS-PAGE analysis was also performed using 4-12% Bis-Tris gels under reducing and non-reducing conditions. Anti-human CEA IgG / AE2P (scFv) 2 bispecific antibody (2+2, Fig.13 The heavy chain and light chain sequences of form 1) are shown in SEQ ID NOs: 61 and 62, respectively.

[0217] In order to evaluate the combination of anti-human CEA IgG / AE2P (scFv) 2 bispecific antibodies, flow cytometry analysis of primary mouse T cells and target mouse C51 colon cancer cells (C51.CEA) expressing human CEA was performed by staining with 50nM bispecific antibodies. First, cells were blocked for non-specific binding for 30 minutes in FACS buffer (PBS, with 2% FBS and 2mM EDTA). After incubation for 1 hour with anti-human CEA IgG / AE2P (scFv) 2 bispecific antibodies, cells were washed twice with FACS buffer (PBS, with 2% FBS and 2mM EDTA), and PE-conjugated anti-human secondary antibodies were added to detect fluorescence shifts in the case of binding. Zombie NIR is used to distinguish live cells from dead cells.

[0218] The anti-human CEA IgG / AE2P(scFv)2 bispecific antibody had good purity ( Fig.17 A and Fig.17 B). Binding of the bispecific antibody to both primary mouse T cells and target cells expressing human CEA (C51.CEA) was confirmed by flow cytometry ( Fig.17 C and Fig.17 D).

[0219] Example 11 - Anti-CD28 IgG / anti-CEA (scFv) 2 bispecific antibody ( Fig.13 The clones of form 2) are Levy

[0220] Cloning the gene of AE2P IgG / anti-human CEA (scFv) 2 bispecific antibody in the form of IgG-(scFv) 2 (2+2), wherein IgG is in the form of IgG4 ( Fig.13 Form 2), expressed using transient gene expression (TGE) in CHO-S, purified by protein A affinity chromatography, dialyzed and stored in PBS. The bispecific antibodies were characterized by size exclusion chromatography using a S200i 10 / 300GL column on FPLC. SDS-PAGE analysis was also performed using 4-12% Bis-Tris gels under reducing and non-reducing conditions. Fig.13 The heavy chain and light chain sequences of form 2) are shown in SEQ ID NOs: 63 and 64, respectively.

[0221] To evaluate the binding of AE2P IgG / anti-human CEA (scFv) 2 bispecific antibodies, flow cytometry analysis of primary mouse T cells and target mouse C51 colon cancer cells expressing human CEA (C51.CEA) was performed by staining with 50nM of bispecific antibodies. First, cells were blocked for nonspecific binding in FACS buffer (PBS, 2% FBS and 2mM EDTA) for 30 minutes. After incubation with AE2P IgG / anti-human CEA (scFv) 2 bispecific antibodies for 1 hour, cells were washed twice with FACS buffer, and PE-conjugated anti-human secondary antibodies were added to detect fluorescence shifts in the case of binding. Zombie near infrared (NIR) was used to distinguish live cells from dead cells.

[0222] The AE2P IgG / anti-human CEA (scFv) 2 bispecific antibody had good purity ( 10.3 mmol / l) as demonstrated by the single peak observed by SEC and the expected molecular weight under reducing and non-reducing conditions when analyzed by SDS-PAGE. Fig.18 A and Fig.18 B). Flow cytometry confirmed the binding of the bispecific antibody to both primary mouse T cells and to target cells expressing human CEA (C51.CEA). Fig.18 C and Fig.18 D).

[0223] Sequence Listing

[0224] SEQ ID NO: 1—Amino acid sequence of the extracellular domain of human CD28

[0225] NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP

[0226] SEQ ID NO:2—Amino acid sequence of the mouse CD28 extracellular domain

[0227] NKILVKQSPLLVVDSNEVSLSCRYSYNLLAKEFRASLYKGVNSDVEVCVGNGNFTYQPQFRSNAEFNCDGDFDNETVTFR LWNLHVNHTDIYFCKIEFMYPPPYLDNERSNGTIIHIKEKHLCHTQSSPKL

[0228] SEQ ID NO:3—Amino acid sequence of AE2P CDR1 VH

[0229] GFTFSSYAMS

[0230] SEQ ID NO:4-AMINO ACID SEQUENCE OF AE2P CDR2 VH

[0231] AISGSGGSTYYADSVKG

[0232] SEQ ID NO:5—Amino acid sequence of AE2P CDR3 VH

[0233] RYIAFDY

[0234] SEQ ID NO:6—Amino acid sequence of AE2P CDR1 VL

[0235] RASQSISSYLN

[0236] SEQ ID NO:7-AMINO ACID SEQUENCE OF AE2P CDR2 VL

[0237] AASSLQS

[0238] SEQ ID NO:8—Amino acid sequence of AE2P CDR3 VL

[0239] QQGGMPPDT

[0240] SEQ ID NO:9—Amino acid sequence of AE2P VH domain

[0241] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRYIAFDYWGQGTLVTVSS

[0242] SEQ ID NO: 10—Amino acid sequence of AE2P VL domain

[0243] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGGMPPDTFGQGTKVEIK

[0244] SEQ ID NO:11 - Amino acid sequence of AE2P antibody molecule in scFv format

[0245] The linker sequences are underlined.

[0246] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRYIAFDYWGQGTLVTVSS GGGGSGGGGSGGGGDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGGMPPDTFGQGTKVEIK

[0247] SEQ ID NO:12—Amino acid sequence of the linker between VH and VL in AE2P scFv

[0248] GGGGSGGGGGSGGGG

[0249] SEQ ID NO: 13 - Amino acid sequence of AE2P heavy chain in IgG1 format

[0250] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRYIAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0251] SEQ ID NO:14—Amino acid sequence of AE2P light chain

[0252] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGGMPPDTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0253] SEQ ID NO:15 - Amino acid sequence of AE2P antibody molecule in SIP form

[0254] The linker sequence has been underlined.

[0255] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRYIAFDYWGQGTLVTVSS GGGGSGGGGSGGGG DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGGMPPDTFGQGTKVEIK SGGSGG PRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFICRAVHEAASPSQTVQRAVSVNPESSRRGGC

[0256] SEQ ID NO:16—Linker between scFv and the εS2_CH4 domain of the SIP format

[0257] SGGSGG

[0258] SEQ ID NO: 17 - Amino acid sequence of the murine IgG2a form of the AE2P heavy chain

[0259] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRYIAFDYWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0260] SEQ ID NO: 18 - Amino acid sequence of the murine IgG2a form of the AE2P light chain

[0261] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGGMPPDTFGQGTKVEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0262] SEQ ID NO: 19 - Amino acid sequence of AE2P antibody in diabody format (Db)

[0263] The linker sequences are underlined.

[0264] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRYIAFDYWGQGTLVTVSS GGSGGDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGGMPPDTFGQGTKVEIK

[0265] SEQ ID NO:20—Amino acid sequence of AE2P antibody in single-chain diabody format (scDb)

[0266] The linker sequence has been underlined.

[0267] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRYIAFDYWGQGTLVTVSS GGSGG DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGGMPPDTFGQGTKVEIK DIGGGSGGGGSGGGG EEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRYIAFDYWGQGTLVTVSS GGSGG DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGGMPPDTFGQGTKVEIK

[0268] SEQ ID NO:21 - Amino acid sequence of the linker between VH and VL in AE2P Db and AE2P scDb

[0269] GGSGG

[0270] SEQ ID NO:22—Amino acid sequence of AE2P heavy chain in IgG4 format

[0271] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRYIAFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0272] SEQ ID NO:23-AE2P-7NP2BiTE TM The amino acid sequence

[0273]

[0274] The linker sequences are underlined.

[0275]

[0276] SEQ ID NO:24-L19-OKT3 BiTE TM The amino acid sequence

[0277]

[0278] The linker sequences are underlined.

[0279]

[0280] SEQ ID NO:25-AE2P-7NP2 BiTE TM Amino acid sequence of AE2P antibody molecule in scFv form

[0281] AE2P VH– Connectors -AE2P VL

[0282] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRYIAFDYWGQGTLVTVSS GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGGMPPDTFGQGTKVEIK

[0283] SEQ ID NO:26-AE2P-7NP2 BiTE TM Amino acids in the linker between VH and VL in AE2P scFv sequence

[0284] GGGGSGGGGSGGGGS

[0285] SEQ ID NO:27 - amino acid sequence of TGN1412 heavy chain (such as SEQ ID NO: 8709414) ID NO:42 in Public

[0286] MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0287] SEQ ID NO:28 - amino acid sequence of TGN1412 light chain (such as SEQ ID NO: 8709414) ID NO:44 in Public

[0288] MGWSCIILFLVATATGVHSDIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0289] SEQ ID NO:29-59 corresponds to Fig.11 Peptides 1-31 used in the peptide array in B.

[0290] SEQ ID NO:60—Anti-CD3 (PUB4) / anti-EDB (L19) BITE TM

[0291]

[0292] The linker sequence has been underlined.

[0293]

[0294] SEQ ID NO:61-Anti-human CEA Amino acid sequence of the heavy chain of IgG / AE2P(scFv)2(2+2) (Format 1)

[0295]

[0296] SEQ ID NO:62—Amino acid sequence of the light chain of anti-human CEA IgG / AE2P (scFv) 2 (2+2) (version 1)

[0297] Anti-human CEA VL- CL

[0298] EIVLTQSPGTLSLSPGERATLSCRASQSVSQNHLAWYQQKPGQAPRLLIYLASRRHTGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQSGRVPWTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0299] SEQ ID NO:63—Amino acid sequence of the heavy chain of AE2PIgG / anti-human CEA (scFv) 2 (2+2) (version 2)

[0300]

[0301]

[0302] SEQ ID NO:64-AE2P Amino acid sequence of the light chain of IgG / anti-human CEA (scFv) 2 (2+2) (Format 2)

[0303] AE2P VL- CL

[0304] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGGMPPDTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0305] SEQ ID NO:65 - Amino acid sequence of AE2P antibody in single-chain diabody format (scDb)

[0306] The linker sequence has been underlined.

[0307] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRYIAFDYWGQGTLVTVSS GGSGG DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGGMPPDTFGQGTKVEIK DIGGGSGGGGSGGGG EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRYIAFDYWGQGTLVTVSS GGSGG DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGGMPPDTFGQGTKVEIK

[0308] SEQ ID Amino acid sequence of NO:66-CD28-Fc fusion protein (also referred to as “CD28-ECD” or “CD28-Fc”)

[0309] Extracellular domain of human CD28 (black), AviTag TM(black), x6 histidine tag (black) and the Fc portion of human IgG1 (underlined).

[0310]

[0311] References

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Claims

1. An antibody molecule that binds to human CD28 and human CTLA-4, wherein the antibody molecule comprises a VH domain and a VL domain, the VH domain comprises a set of complementary determining regions HCDR1, HCDR2 and HCDR3, and the VL domain comprises a set of complementary determining regions LCDR1, LCDR2 and LCDR3, wherein: The HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs 3, 4 and 5, respectively, and The LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences shown in SEQ ID NOs 6, 7 and 8, respectively.

2. The antibody molecule according to claim 1, wherein the VH domain comprises the amino acid sequence shown in SEQ ID NO:9 and / or the VL domain comprises the amino acid sequence shown in SEQ ID NO:

10.

3. The antibody molecule of claim 1 or claim 2, wherein the antibody molecule further binds to mouse CD28.

4. The antibody molecule according to any one of claims 1 to 3, wherein the antibody molecule is human or humanized.

5. The antibody molecule according to any one of claims 1 to 4, wherein the antibody molecule comprises or consists of a single-chain Fv (scFv), a diabody (Db), a single-chain diabody (scDb), a small immune protein (SIP), an IgG1 molecule, an IgG2a molecule or an IgG4 molecule.

6. The antibody molecule according to claim 5, wherein the antibody molecule comprises the amino acid sequence shown below: SEQ ID NO: 11; SEQ ID NO: 15; SEQ ID NO: 19; SEQ ID NO:65; or SEQ ID NO: 25; Or wherein the antibody molecule comprises the heavy chain amino acid sequence shown in SEQ ID NO:22, SEQ ID NO:17 or SEQ ID NO:13 and the light chain amino acid sequence shown in SEQ ID NO:

14.

7. A conjugate comprising the antibody molecule according to any one of claims 1 to 6 and a pro-inflammatory agent or a radioactive isotope.

8. The antibody molecule of claims 1 to 6, wherein the antibody further comprises a second antigen binding site that binds a tumor-associated antigen, optionally wherein the tumor-associated antigen is selected from the group consisting of: fibroblast activation protein (FAP), ED-A, ED-B or IIICS isoforms of fibronectin, CAIX, CEA, mucin-16, PSMA and A, A1, A2, B, C or D isoforms of tenascin C.

9. The antibody molecule according to claim 8, wherein the antibody molecule is: (i) bispecific T cell engagers; (ii) scDb-scFv; (iii) IgG-(scFv); or (iv) IgG-(scFv)2.

10. The antibody molecule according to claim 9, wherein: The antibody molecule is a bispecific T cell engager and comprises the amino acid sequence shown in SEQ ID NO:23; The antibody molecule is IgG-(scFv)2 and comprises the heavy chain amino acid sequence shown in SEQ ID NO:61 and the light chain amino acid sequence shown in SEQ ID NO:62; or The antibody molecule is IgG-(scFv)2 and comprises the heavy chain amino acid sequence shown in SEQ ID NO:63 and the light chain amino acid sequence shown in SEQ ID NO:

64.

11. An antibody molecule or conjugate according to any one of claims 1 to 10 for use in a method of treating the human or animal body by therapy.

12. The antibody molecule or conjugate according to any one of claims 1 to 10 for use in a method of treating cancer in a patient.

13. The antibody molecule for use according to claim 11 or claim 12, wherein the method further comprises administering to the patient a second therapeutic agent, wherein the second therapeutic agent is selected from: (i) chemotherapy; (ii) radiotherapy; (iii) checkpoint inhibitors; (iv) an agonist that binds to GITR, OX40 or 4-1BB; (v) a co-stimulatory anti-CD28 bispecific antibody; or (vi) Antibodies that bind to human CD3.

14. A nucleic acid molecule or an expression vector encoding the antibody molecule or conjugate according to any one of claims 1 to 10, or a host cell comprising the nucleic acid or the expression vector.

15. A method for producing an antibody molecule or a conjugate according to any one of claims 1 to 10, the method comprising culturing a host cell according to claim 14 under conditions for expressing the antibody or conjugate, and optionally further isolating and / or purifying the antibody molecule or conjugate after expression.

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