Small shedding blocker

Abstract

An agent of less than 100 kilodaltons is provided that binds to a membrane immunoreceptor on the surface of a cell and inhibits proteolytic cleavage of the immunoreceptor. A method of treating cancer and enhancing immunotherapy comprising administering the agent is also provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 954,802 filed on December 30, 2019, U.S. Provisional Patent Application No. 62 / 942,240 filed on December 2, 2019, and U.S. Provisional Patent Application No. 62 / 818,351 filed on March 14, 2019, the entire contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The invention belongs to the field of immunomodulation and immunotherapy. Background Art

[0004] The adaptive immune system plays a key role in the regulation and protection against pathogens and cancer cells, primarily through the coordinated stimulation of antigen-specific helper CD4+ and cytotoxic CD8+ T cells. The persistent and continuous activation of T cells by antigen presenting cells (APCs) involves i) the engagement of the T cell receptor (TCR) with peptides presented by the major histocompatibility complex (MHC) on the APC; and ii) the co-stimulatory CD28 receptor on T cells that binds to the B7-1 (CD80) and B7-2 (CD86) ligands also expressed by the APC. The biological consequences of CD28 co-stimulation are numerous and include control of the T cell cycle, expansion, differentiation, and amplification of TCR stimulation by lowering the threshold required to achieve immune effector functions.

[0005] Unlike the activating co-stimulatory molecule CD28, the structural homolog, cytotoxic T lymphocyte-associated 4 (CTLA-4), is an inhibitory co-stimulatory receptor whose membrane expression is driven by the triggering of CD28. Both CTLA-4 and CD28 are type I transmembrane proteins. Their extracellular parts consist of a group V immunoglobulin superfamily (Ig-V) domain, which is covalently linked by cysteine ​​residues located outside the IgV domain near the transmembrane region. Despite the similarities, CTLA-4 and CD28 differ in affinity and quaternary structural arrangement. CTLA-4 was found to have a higher binding affinity for B7 molecules, and a different dimerization mode from CD28 resulted in different stoichiometric binding to the shared ligand. CD28 exhibits a monovalent binding stoichiometry, while CTLA-4 interacts in a bivalent manner. Therefore, CTLA-4 binds to B7 molecules with much higher affinity and avidity than CD28, and thereby downregulates T cell responses and contributes to the initiation of antigen-specific tolerance.

[0006] It has been shown that some co-stimulatory molecules have several physiological forms.Except for the membrane-bound form, a soluble form expressed in immature immune cells has been described, increasing the complexity of T cell biology.The soluble form of CD28 (sCD28) has been attributed to the gene product of alternative splicing.Splicing events lead to frameshifting, and the result is that two glutamic acid residues are added after the 137th glycine before translation termination.The final product lacks the entire transmembrane and cytoplasmic region, and importantly lacks the cysteine ​​residues at position 141, which mediates the disulfide bond of dimer CD28 (Magistrelli G., BiochemBiophyRes Commun, 1999).The biological function and anti-receptor binding of monomeric CD28 soluble form have been checked (Hebbar, M., Clin Exp Immunol, 2004) and it is shown that T cell proliferation is also inhibited. Likewise, in the case of dimeric sCD28, a regulatory role of inhibiting T cell function by binding to B7 molecules has been suggested (Sun, Z., Centr Eur J Immunol, 2014; Hebbar, M., Clin Exp Immunol, 2004). Of note, an increase in the number of sCD28 molecules in the serum of patients with autoimmune disorders has been reported (Wong, CK, Rheumatol, 2005; Hamzaoui, K., Clin Exp Rheumatol, 2005; Hebbar, M., Clin Exp Immunol, 2004; Sun, Z., Clin Immunol, 2014). The exact origin of sCD28 is controversial. Using an in vitro model of T cell activation, reflecting the persistent inflammatory state of T cells in autoimmune patients, it has been shown that during T cell activation, the transcription of the optional soluble form is inhibited, and only the full-length membrane form of CD28 is evident, while the amount of sCD28 in the culture increases (Hebbar, M., Clin Exp Immunol, 2004). This phenomenon leads to the suggestion that the effective shedding of the membrane form of CD28 is the reason for the increase of the soluble molecule in the serum, however, this has yet to be confirmed. In the past, effective shedding during T cell activation was described as a regulatory mechanism that counteracts continuous activation by proteolysis of adhesion molecules.

[0007] While CTLA-4 limits the amplitude of early T cell responses, another inhibitory receptor, PD-1, inhibits T cell function in the periphery. The expression of PD-1 increases during T cell activation, and its known ligands are B7 family homologs: B7-H1 (PD-L1) and B7-H2 (PD-L2). These homologs are present on APCs and cancer cells and drive activated T cells into a state of cellular anergy, resulting in a weakened immune response. Therefore, targeted therapies for CTLA-4 and PD-1 / PD-L1 axes have shown clinical activity in a variety of cancer types. Recently, studies have shown that the signaling pathway of CD28 is targeted and inhibited by PD-1 (Hui, E., Science, 2017) and concomitantly for the conduct of effective PD-1 therapy, a complete active CD28 / B7 axis is essential (Kamphorst, AO, Science, 2017).

[0008] However, not all patients respond to PD-1-based immunotherapy, or immunotherapy in general, and those who do often relapse. Therefore, methods and molecules that can improve the ability of a patient's immune cells to attack cancer are greatly needed. Summary of the invention

[0009] The present invention provides an agent of less than 100 kilodaltons that binds to membrane CD28 (mCD28) on the cell surface and inhibits proteolytic cleavage of mCD28. Also provided is a method for treating and preventing cancer and improving PD-1 / PD-L1-based immunotherapy, which comprises administering the agent.

[0010] According to a first aspect, there is provided an agent that binds membrane CD28 (mCD28) on the surface of a cell and inhibits proteolytic cleavage of mCD28, wherein the agent is less than 100 kilodaltons (kDa).

[0011] According to another aspect, a method of reducing the level of soluble CD28 (sCD28) in a subject in need thereof is provided, the method comprising administering an agent of the invention.

[0012] According to another aspect, there is provided a method of treating and / or preventing cancer in a subject in need thereof, the method comprising administering an agent of the present invention.

[0013] According to another aspect, a method of improving PD-1 and / or PD-L1 based immunotherapy in a subject in need thereof is provided, the method comprising administering an agent of the invention.

[0014] According to another aspect, a method for producing an agent for inhibiting proteolytic cleavage of mCD28 on the surface of a cell is provided, comprising at least one of the following steps:

[0015] a. obtaining an agent that binds to the extracellular domain of CD28 or a fragment thereof, wherein the agent is less than 100 kDa;

[0016] b. testing the obtained reagent for binding to mCD28 on the cell surface; and

[0017] c. Selecting a reagent that binds to cell surface mCD28;

[0018] and

[0019] d. Cultivating a host cell comprising one or more vectors comprising a nucleic acid sequence encoding an agent, wherein the nucleic acid sequence is a nucleic acid sequence of an agent selected by:

[0020] i. obtaining an agent that binds to the extracellular domain of CD28 or a fragment thereof, wherein the agent is less than 100 kDa;

[0021] ii. testing the obtained reagent for binding to mCD28 on the cell surface; and

[0022] iii. selecting a reagent that binds to cell surface mCD28;

[0023] This generates an agent that inhibits proteolytic cleavage of mCD28 on the cell surface.

[0024] According to another aspect, there is provided an agent produced by the method of the present invention.

[0025] According to another aspect, there is provided a pharmaceutical composition comprising an agent of the invention and a pharmaceutically acceptable carrier, excipient or adjuvant.

[0026] According to another aspect, a method of treating and / or preventing cancer, improving PD-1 and / or PD-L1 based immunotherapy, or reducing sCD28 levels in a subject in need thereof is provided, the method comprising administering a pharmaceutical composition of the present invention.

[0027] According to another aspect, a kit comprising at least one agent of the invention is provided.

[0028] According to some embodiments, the agent is selected from the group consisting of an antigen-binding fragment of an antibody, a Fab fragment, a single chain antibody, a single domain antibody, a small molecule, and a peptide that specifically binds to CD28.

[0029] According to some embodiments, the agent is less than 50 kDa.

[0030] According to some embodiments, the single domain antibody is a camelid antibody or a shark antibody.

[0031] According to some embodiments, the camelid antibody comprises three CDRs, wherein:

[0032] CDR1 comprises the amino acid sequence set forth in SEQ ID NO:33 (INAMG), CDR2 comprises the amino acid sequence set forth in SEQ ID NO:34 (AISGGGDTYYADSVKG), and CDR3 comprises the amino acid sequence set forth in SEQ ID NO:35 (DLYGSDYWD);

[0033] CDR1 comprises the amino acid sequence set forth in SEQ ID NO:36 (INAMA), CDR2 comprises the amino acid sequence set forth in SEQ ID NO:37 (AITSSGSTNYANSVKG), and CDR3 comprises the amino acid sequence set forth in SEQ ID NO:38 (DEYGSDYWI); or

[0034] CDR1 includes the amino acid sequence set forth in SEQ ID NO:33 (INAMG), CDR2 includes the amino acid sequence set forth in SEQ ID NO:39 (AITSGGSTNYADSVKG), and CDR3 includes the amino acid sequence set forth in SEQ ID NO:40 (DLYGEDYWI).

[0035] According to some embodiments, the camelid antibody comprises a sequence selected from the group consisting of:

[0036] a.EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVTVSS (SEQ ID NO: 30);

[0037] and

[0038] c. QVQLVESGGGLVQAGGSLRLSCAASGSIFSINAMGWYRQAPGKQRERVAAITSGGSTNYADSVKGRFTISRDNAKNTVYLQMNNLEPRDAGVYYCVVDLYGEDYWIWGQGTQVTVSS (SEQ ID NO: 32).

[0039] According to some embodiments, the reagent comprises three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein:

[0040] CDR-H1 includes the amino acid sequence described in SEQ ID NO:17 (GFTFSSYYMS), CDR-H2 includes the amino acid sequence described in SEQ ID NO:18 (TISDGGDNTYYAGTVTG), CDR-H3 includes the amino acid sequence described in SEQ ID NO:19 (IHWPYYFDS), CDR-L1 includes the amino acid sequence described in SEQ ID NO:20 (RASSSVSYMN), CDR-L2 includes the amino acid sequence described in SEQ ID NO:21 (ATSDLAS), and CDR-L3 includes the amino acid sequence described in SEQ ID NO:22 (QQWSSHPPT).

[0041] According to some embodiments, the agent is humanized.

[0042] According to some embodiments, the agent is not a CD28 agonist.

[0043] According to some embodiments, the agent is not a CD28 antagonist.

[0044] According to some embodiments, the agent neither degrades mCD28 nor inhibits mCD28-mediated immune cell activation.

[0045] According to some embodiments, the antigen-binding fragment of the antibody does not induce antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0046] According to some embodiments, the agent binds within the stalk region of CD28.

[0047] According to some embodiments, the stem region comprises the amino acid sequence GKHLCPSPLFPGPSKP (SEQ ID NO: 9) or KGKHLCPSPLFPGPS (SEQ ID NO: 27).

[0048] According to some embodiments, the stem region consists of the amino acid sequence HVKGKHLCPSPLFPGPSKP (SEQ ID NO: 10).

[0049] According to some embodiments, the reagent binds at least one protease at the cleavage site.

[0050] According to some embodiments, the agent inhibits protease cleavage by at least one protease.

[0051] According to some embodiments, the at least one protease is at least one metalloprotease.

[0052] According to some embodiments, the at least one metalloprotease is MMP-2, MMP-13, or a combination thereof.

[0053] According to some embodiments, the subject has cancer.

[0054] According to some embodiments, the cancer is selected from melanoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, renal cancer, gastric cancer, and colorectal cancer.

[0055] According to some embodiments, the cancer is selected from melanoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, and colorectal cancer.

[0056] According to some embodiments, the methods do not reduce mCD28 or reduce mCD28-mediated immune cell activation.

[0057] According to some embodiments, the subject's blood comprises at least 5 ng / ml sCD28 prior to administration.

[0058] According to some embodiments, obtaining is obtaining an agent that is less than 50 kDa, and wherein the obtained agent is less than 50 kDa.

[0059] According to some embodiments, the method further comprises testing the agent for its ability to block cleavage by a protease of mCD8 on the cell surface.

[0060] According to some embodiments, the protease is selected from MMP-2 and MMP-13.

[0061] According to some embodiments, obtaining the reagent comprises at least one of the following steps:

[0062] a. Immunizing sharks or camelids using the CD28 extracellular domain or a fragment thereof, and collecting antibodies from the immune organisms; and

[0063] b. Screening a library of agents that bind to the CD28 extracellular domain or a fragment thereof and selecting a binding agent.

[0064] According to some embodiments, the CD28 extracellular domain or fragment thereof is dimeric or monomeric.

[0065] According to some embodiments,

[0066] a. collecting antibodies comprising extracting B cells from the spleen of an immunized shark or camelid; or

[0067] b. Selecting a binding agent includes sequencing the selected agent and generating a recombinant form of the agent from the sequence.

[0068] According to some embodiments, the method further comprises measuring mCD28 downstream signaling in the presence of the obtained agent and selecting at least one agent that neither substantially agonizes nor substantially antagonizes mCD28 signaling.

[0069] According to some embodiments, the kit further comprises at least one of the following:

[0070] a. Anti-PD-1 and / or PD-L1 immunotherapy; and

[0071] b. Describe the labeling of the reagent of the present invention for use in PD-1 and / or PD-L1-based immunotherapy.

[0072] From the detailed description given below, other embodiments and the entire scope of application of the present invention will become apparent. However, it should be understood that although the detailed description and specific examples show the preferred embodiments of the present invention, they are only given as illustrations, because various changes and modifications within the spirit and scope of the present invention are apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 . Soluble CD28 is produced during PBMC stimulation and counteracted by the addition of protease inhibitors (PIs) Bar graph of the amount of soluble CD28 in cultures of PBMCs stimulated with SEB (0.5 ng / mL, left) or CMV peptide (0.5 μg / mL, right) was quantified by human CD28 ELISA (top). A cocktail of protease inhibitors was added at the indicated concentrations. Overall health and effector activity were examined by secretion of interferon gamma (bottom).

[0074] Figure 2 . Soluble CD28 is produced during T cell stimulation with PHA and is counteracted by the addition of protease inhibitors. remove Bar graphs of Jurkat cells (upper left) or isolated human CD4 T cells (upper right) stimulated with increasing concentrations of PHA (1-4 μg / mL, top panel) in the presence of a protease inhibitor cocktail at a fixed concentration (2 μM). In another setup, a fixed PHA concentration was used to stimulate Jurkat T cells (1 μg / mL PHA, lower left) or human CD4 T cells (2 μg / mL PHA, lower right), and the concentration of the protease inhibitor cocktail was titrated (0.5-2 μM). The concentration of human CD28 in the supernatant was quantified using a standard sandwich ELISA.

[0075] Figure 3A-3B . Specific ADAM-10 and ADAM-17 inhibitors eliminate soluble CD28 accumulation but does not suppress their viability(3A-B) Bar graphs of human PMBCs stimulated with SEB (1 ng / mL) in the presence of (3A) ADAM-10 specific inhibitor (GI254023X) and (3B) ADAM-17 specific inhibitor (TMI-1) at various concentrations (0.01-1 μM). The viability of cells in the different treatments was assessed using the MTT assay (upper panel). The concentration of human CD28 in the supernatant was quantified using a standard sandwich ELISA (lower panel).

[0076] Figures 4A-4D . Soluble CD28 is produced during PBMC stimulation. (4A) Bar graph of immature dendritic cells mixed with CD3 T cells from the same donor at a ratio of 1:5 without CMV peptide (black bars) or with CMV peptide (dark gray bars). Controls for each cell population alone or with CMV are light gray bars. The concentration of human CD28 in the supernatant was quantified by standard sandwich ELISA. (4B-D) Bar graphs of human PBMCs stimulated with (4B) CMV or (4C) SEB or (4D) SEB for 24 hours in the presence of ADAM-10 and ADAM-17 inhibitors and then transferred to clean culture. Figure 4D Measurements in were taken 120 hours after cell transfer.

[0077] Figure 5 . Soluble CD28 inhibits effector cytokine secretion Bar graph of human PBMCs stimulated with CMV (0.5 μg / mL) without recombinant human CD28 (black bars) or with indicated concentrations of recombinant human CD28 (grey bars). Naive samples without CMV stimulation are indicated by light grey bars. The concentration of human IFNγ in the supernatant was quantified using a standard sandwich ELISA (Biolegend).

[0078] Figure 6 . Soluble CD28 increases IL-6 cytokine secretion Bar graph of human PBMCs stimulated with CMV (0.5 μg / mL) without recombinant human soluble CD28 (black bars) or with indicated concentrations of recombinant human soluble CD28 (grey bars). Naive samples without CMV stimulation are indicated by light grey bars. The concentration of human IL-6 in the supernatant was quantified using a standard sandwich ELISA (Biolegend).

[0079] Figures 7A-7E.(7A) Line graph of human PBMCs stimulated with CMV (0.5 μg / mL) in the presence of recombinant human soluble CD28 (grey triangles) or recombinant human soluble CTLA-4 (black circles) at the indicated concentrations. The concentrations of human IL-6, IFNγ, and IL-4 in the supernatant were quantified using standard sandwich ELISA (Biolegend). The concentrations of human IL-8, IL-12p (40), and IL-10 in the supernatant were quantified using multiplex analysis using the Magpix system (Millipore). (7B) Bar graph of cytokine secretion by autologous monocytes and CD3MLR. Naive samples without CMV stimulation are indicated by light grey bars. CMV alone or with IgG control is indicated by black bars. Increasing concentrations of sCD28 are indicated by dark grey bars. (7C) Line graph of lymphocyte cluster formation by human PBMCs stimulated with SEB in the presence of recombinant human soluble CD28 (grey circles) or with control IgG (grey triangles). (7D) Bar graph of IDO secretion into culture measured by kynurenine ELISA kit from monocytes treated with and without recombinant human sCD28. (7E) Scatter plot of intracellular FACS of IDO in monocytes treated with and without recombinant human sCD28.

[0080] Figures 8A-8C . Soluble CD28 impedes anti-PD1 processing (8A) Bar graph of human PBMCs stimulated for 3 days with SEB (200 ng / mL, left bar) or CMV peptide (0.5 μg / mL, right bar) in the presence of anti-PD1 (MK3475, 5 μg / mL, black bars) or recombinant human soluble CD28 (2 and 10 μg / mL, gray bars) or a combination of both (dashed bars). (8B) Bar graph of cytokine secretion in a monocyte MLR setup, naive - white bars, CMV alone - light gray bars, sCD28 - black bars, MK-3475 - dark gray bars, sCD28 + MK-3475 - checkered bars. Concentrations of human IFNγ, TGFβ, and IL-2 in supernatants were quantified using standard sandwich ELISA (Biolegend). (8C) Histogram of surface PD-L1 (left) and PD-L2 (right) expression in monocytes after incubation with control and sCD28.

[0081] Figures 9A-9C . Soluble CD28 in cancer patients. (9A) Dot plots showing 20 plasma samples from each of 10 cancer indications and healthy donors investigated for the presence of soluble human CD28. Samples containing high levels of soluble CD28 were examined repeatedly with various dilution factors. The concentration of human CD28 in the supernatant was quantified using a standardized sandwich ELISA, internally calibrated to adapt the readings of human plasma samples. (9B) Bar graph of IFNγ secretion from SEB-stimulated PBMCs from cancer patients measured by sandwich ELISA in the presence of sCD28, MK-3475, and a combination of both (sarcoma patient - upper left, renal cancer patient - upper right, and two different head and neck cancer patients - bottom). (9C) Bar graph of cancer cell SCC-25 viability and proliferation alone, in culture with IL-6, in co-culture with monocytes, or in co-culture with monocytes and sCD28.

[0082] Figures 10A-10B (10A) Bar graph of IFNγ of isolated CD3 T cells stimulated with anti-CD3 in the presence of constant CD80-Fc levels and titrated soluble CD28. (10B) Isolated PBMCs stimulated with CMV in the presence of constant sCD28 levels and titrated CD80-Fc.

[0083] Figures 11A-11B .(11A-B) Line graphs of tumor volume of H22 cells inoculated in immunocompetent mice treated with anti-PD-1 antibody, without administration of recombinant mouse CD28 (11A) and with administration of recombinant mouse CD28 (11B).

[0084] Figures 12A-12C.(12A) Line graph showing antigen binding by serial dilution of clone M9 conjugated to BSA CD28 stem region dimer peptide (right) and recombinant human CD28 protein (left). Antigen was immobilized on a maxisorp ELISA plate. A dilution series of clone M9 was performed and bound antibody was detected using donkey anti-mouse IgG (H&L)-HRP and developed using TMB. (12B) Bar graph of ELISA detection of recombinant human sCD28 (left) and sCD28 shed from human PBMC activated with SEB (right). The ELISA used antibody #3 as a positive control (2 μg / mL, gray bars), irrelevant antibody M39 as a negative control (10 μg / mL, dark gray bars), and anti-cleavage antibody M9 (10 μg / mL, black bars). Detection of recombinant CD28 or shed CD28 was performed by using an ELISA kit detection antibody conjugated to HRP (0.5 μg / mL). (12C) Histogram showing the binding of antibody M9 (top) and control antibody CD28.2 (bottom) to human CD28 expressed in mouse HEK293 cells at a fixed concentration of 10 μg / ml (black histogram). Polyclonal mouse IgG was used as a negative control (10 μg / ml) and is depicted in the gray histogram. Detection was accomplished by secondary incubation of AlexaFluor 647-conjugated goat anti-mouse.

[0085] Fig.13 Binding to human CD28 stem region sequences by ELISA. Antigen binding was analyzed by serial dilution of different VHH clones. Biotin-conjugated CD28 stem region dimer peptide used as antigen was immobilized on neutravidin-coated ELISA maxi-sorb plates. Serial dilutions of VHH clones were performed and bound VHHs were detected using anti-His tag-HRP conjugated antibody and developed using TMB.

[0086] Fig.14 . Binding of VHH #2A1 to membrane human CD28. FITC-conjugated VHH clone 2A1 (50 μg / mL, black histogram) and FITC-conjugated isotype control (mIgG, 50 μg / mL, gray histogram) were incubated with HEK cells overexpressing human CD28. Binding was assessed by FACS analysis.

[0087] Fig.15.Anti-CD28 stem region VHH clones do not block ligand binding to membrane CD28. HEK293 cells overexpressing human CD28 were monitored by flow cytometry for CD86-Fc (2 μg / mL) binding using a secondary anti-human Fc antibody conjugated to AlexaFlour 647. Addition of anti-CD28 VHH clones (30 μg / mL, black histogram) to CD86-Fc did not change the size of CD86 binding, while addition of commercial antibody clone CD28.2 (10 μg / mL, upper left panel, black histogram) significantly reduced binding.

[0088] Fig.16 . Evaluation of the agonist effect of anti-CD28 VHH clones. Human isolated CD3 cells were stimulated for 2 days with plate-bound anti-CD3 (OKT3, 2 μg / mL, light grey bars) in the presence of anti-CD28 agonist antibody clone 28.2 (2 μg / mL, dark grey), anti-CD28 stem region VHH or irrelevant VHH clone (20 μg / mL, black bars) used as positive control. The concentration of human IFNγ secreted into the supernatant was quantified using a standard sandwich ELISA (Biolegend).

[0089] Fig.17 . Blocking of MMP-2-mediated cleavage of the human CD28 stem region in vitro by VHH clones. c-Myc-conjugated and biotinylated human CD28 stem region dimer peptide (1 μM) was incubated with 50 ng rhMMP-2 in the presence of MMP-2 inhibitor (TMI-1, 50 nM) M9 Fab or indicated VHH clones at various concentrations (0.4-10 μg / mL) for 5 hours. The mixture was loaded on neutravidin-coated ELISA maxi-sorb plates, followed by extensive washing and detection of intact peptides by anti-cMyc-HRP-conjugated antibodies and developed with TMB.

[0090] Fig.18 Anti-CD28 stem region VHH clones 2A1 and 4A4 inhibit CD28 shedding in HEK cells overexpressing human CD28. Soluble CD28 levels were measured in the culture medium of HEK cells stably expressing human CD28 after 48 hours of incubation. The effect of different treatments with MMP inhibitors (TMI-1, 1 μM, dark grey bars) at various concentrations (3.3-100 μg / mL), negative controls of irrelevant VHHs (upper left panel, black bars) or anti-CD28 stem region VHH clones (black bars) on the amount of soluble CD28 is depicted. Soluble human CD28 levels in the supernatant were quantified using a standard sandwich ELISA (R&D Systems).

[0091] Fig.19Anti-CD28 stem region VHH clones 2A1 and 4A4 inhibit CD28 shedding in isolated CD4 T cells activated by PHA and IL2. Soluble CD28 levels were measured in the culture medium of isolated human CD4 T cells stimulated with 5 μg / mL PHA and 200 IU / mL IL-2 (light grey bars). The effects of different treatments with MMP inhibitors (TMI-1, 1 μM, dark grey bars), negative controls of irrelevant VHHs (upper left, black bars), anti-CD28 stem region VHH clones or Fab formats of antibody M9 clones (black bars) at various concentrations (0.4-50 μg / mL) are depicted on the amount of soluble CD28. The levels of soluble human CD28 in the supernatant were quantified using a standard sandwich ELISA (R&D Systems).

[0092] Fig. 20 . Anti-CD28 stem region VHH clones 2A1 and 4A4 inhibit CD28 shedding in PBMCs activated by superantigens. The level of soluble CD28 was measured in the culture medium of isolated PBMCs stimulated with 1 ng / mL SEB (light gray bars). The effect of different treatments of MMP inhibitors (TMI-1, 1 μM, dark gray bars), negative controls of irrelevant VHHs (upper left figure, black bars), anti-CD28 stem region VHH clones or M9 cloned Fab formats (black bars) on the amount of soluble CD28 are depicted at various concentrations (0.4-50 μg / mL). The level of soluble human CD28 in the supernatant was quantified using a standard sandwich ELISA (R&D Systems).

[0093] Fig.21 Evaluation of the antagonist effect of anti-CD28 VHH clones. Human isolated CD3 cells were stimulated for 24 h with plate-bound anti-CD3 (OKT3, 2 μg / mL, light grey bars) in the presence of recombinant CD80-Fc protein (5 μg / mL, dark grey bars) used as ligand for CD28 co-stimulation. An irrelevant VHH clone (upper left) or anti-CD28 stem region VHH was added at various concentrations (3.75-30 μg / mL, black bars). The concentration of human IL-2 in the supernatant was quantified using a standard sandwich ELISA (Biolegend).

[0094] Fig. 22In vitro blocking activity of VHH clone 2A1 on cleavage of the stem region of human CD28 by MMP-13. c-Myc and biotinylated human CD28 stem region dimer peptide (1 μM) were incubated with 50 ng rhMMP-13 (light gray bars) for 5 hours in the presence of MMPi (TMI-1, 50 nM, dark gray bars), irrelevant VHH clones (black bars in the left panel), or VHH clone 2A1 (black bars in the right panel) at various concentrations (0.62-10 μg / mL). The mixture was loaded on ELISA maxi-sorb plates coated with neutravidin, followed by extensive washing and detection of intact peptides by anti-cMyc-HRP conjugated antibodies and development with TMB.

[0095] Fig.23 . Anti-CD28 stem region VHH clones 2A1, 4A1 and 4A4 specifically bind to the MMP cleavage site of human CD28. Comparison of sequence-specific binding of VHH clones to the human CD28 stem region WT sequence or to the L145 mutation by direct ELISA. Biotin-conjugated wild-type or L145K CD28 stem region dimer peptides were immobilized on neutravidin-coated ELISA maxi-sorb plates. Serial dilutions of VHH clones (0.2-5 μg / mL) and irrelevant VHH clones (upper left) were performed and bound VHHs were detected using an anti-His tag-HRP conjugated antibody and developed using TMB. DETAILED DESCRIPTION

[0096] In some embodiments, the present invention provides an agent of less than 100 kilodaltons (kDa) that binds to membrane CD28 (mCD28) on the cell surface and inhibits protease cleavage of mCD28. Also provided is a method for treating cancer in a subject, improving PD-1 / PD-L1-based immunotherapy, and reducing sCD28 levels, comprising administering an agent of the present invention. The agents and methods of the present invention are based on the following surprising discovery: full-size antibodies against the cleavage site of mCD28 are too large to approach the proximal region of the membrane, and therefore cannot inhibit shedding. Instead, a smaller agent specific for mCD28 on the cell surface is required. Further, a large number of cancer patients have elevated sCD28 levels in their bloodstream, which is caused by sCD28 shedding. The sCD28 acts as an immunosuppressant, and therefore the reduction in shedding has the dual benefits of reducing inhibition by sCD28 and increasing immune activation by mCD28 signaling. Further, it was unexpectedly found that sCD28 can inhibit PD-1 / PD-L1-based immunotherapy.

[0097] Reagents

[0098] According to a first aspect, there is provided an agent that binds membrane CD28 (mCD28) and inhibits proteolytic cleavage of mCD28.

[0099] In some embodiments, mCD28 is on the cell surface. In some embodiments, mCD28 is in the membrane. In some embodiments, the agent is not a full-size antibody. In some embodiments, the agent is not an IgG. In some embodiments, the agent is less than 100 kilodaltons (kDa). In some embodiments, the agent is less than 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20 or 15 kDa. Each possibility represents a separate embodiment of the present invention. In some embodiments, the agent is less than 50 kDa. In some embodiments, the agent is less than 25 kDa. In some embodiments, the agent is less than 20 kDa. In some embodiments, the agent is less than 15 kDa.

[0100] In some embodiments, CD28 is mammalian CD28. In some embodiments, CD28 is human CD28. In some embodiments, human CD28 comprises or consists of the following amino acid sequence: MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 1). In some embodiments, mature CD28 lacks a signal peptide and comprises the sequence: NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 2).

[0101] In some embodiments, the DNA coding sequence encoding full-length human CD28 comprises the sequence: ATGCTCAGGCTGCTCTTGGCTCTCAACTTATTCCCTTCAATTCAAGTAACAGGAAACAAGATTTTGGTGAAGCAGTCGCCCATGCTTGTAGCGTACGACAATGCGGTCAACCTTAGCTGCAAGTATTCCTACAATCTCTTCTCAAGGGAGTTCCGGGCATCCCTTCACAAAGGACTGGATAGTGCTGTGGAAGTCTGTGTTGTATATGGGAATTACTCCCAGCAGCTTCAGGTTTACTCAAAAACGGGGTTCAACTGTGATGGGAAATTGGGCAATGAATCAGTGACATTCTACCTCCAGAATTTGTATGTTAACCAAACAGATATTTACTTCTGCAAAATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCTGA (SEQ ID NO:3).

[0102] As used herein, sCD28 refers to any CD28 fragment or variant that does not include a transmembrane domain and therefore cannot be integrated in a membrane. In some embodiments, the CD28 transmembrane domain includes the amino acid sequence FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 4). In some embodiments, sCD28 is not membrane-bound. In some embodiments, sCD28 is in solution. In some embodiments, sCD28 is CD28 in blood. In some embodiments, sCD28 is CD28 in TME. In some embodiments, sCD28 is CD28 in body fluids. In some embodiments, sCD28 lacks exon 3 of CD28. In some embodiments, sCD28 is a splice variant produced by alternative splicing of exon 3 of splicing away CD28. In some embodiments, sCD28 is a splice product from membrane CD28 (mCD28). In some embodiments, sCD28 is a truncated CD28. In some embodiments, sCD28 lacks the cytoplasmic domain of full-length CD28. In some embodiments, sCD28 is a dimeric sCD28. In some embodiments, sCD28 is a monomeric sCD28. In some embodiments, sCD28 is not a splice variant produced by alternative splicing CD28. In some embodiments, alternative splicing splices out exon 3 of CD28. In some embodiments, sCD28 comprises the amino acid sequence: MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGEE (SEQ ID NO: 5). In some embodiments, sCD28 consists of the amino acid sequence of SEQ ID NO: 5. In some embodiments, sCD28 lacks a signal peptide and comprises the sequence: NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGEE (SEQ ID NO: 6). In some embodiments, sCD28 consists of the amino acid sequence of SEQ ID NO: 6.In some embodiments, sCD28 comprises the amino acid sequence: MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSP (SEQ ID NO: 48). In some embodiments, sCD28 consists of the amino acid sequence of SEQ ID NO: 48. In some embodiments, sCD28 lacks a signal peptide and comprises the sequence: NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSP (SEQ ID NO: 49). In some embodiments, sCD28 consists of the amino acid sequence of SEQ ID NO:49.

[0103] In some embodiments, the DNA coding sequence encoding human sCD28 comprises the sequence: (SEQ ID NO: 7).

[0104] The effects of sCD28 on immune cells are well known in the art and include, as non-limiting examples, immune cell induction of anti-inflammatory cytokines such as IL-10 or TGFβ, immune cell expression of indoleamine 2,3-dioxygenase (IDO), and immune cell downregulation of pro-inflammatory cytokines such as IL-2 or IFN-γ. In some embodiments, the agent inhibits proteolytic cleavage of membrane CD28 including inhibiting the production of sCD28. In some embodiments, inhibiting the production of sCD28 includes inhibiting the effects of sCD28 on immune cells.

[0105] As used herein, inhibition of protease cleavage refers to any reduction of the protease cleavage of mCD28. In some embodiments, inhibition is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% shear reduction. Each possibility represents a separate embodiment of the present invention. In some embodiments, inhibition of protease cleavage maintains the level of mCD28 to immune cells. In some embodiments, inhibition of protease cleavage increases the level of mCD28 to immune cells. In some embodiments, inhibition of protease cleavage maintains the mCD28 level sufficient to immunostimulate.

[0106] In some embodiments, the reduction of protease cleavage is a reduction in the cleavage of at least one protease. In some embodiments, the reduction of protease cleavage is a reduction in the cleavage of at least one metalloprotease. In some embodiments, the metalloprotease MMP-2, ADAM10, ADAM17 or a combination thereof. In some embodiments, the metalloprotease is MMP-2, ADAM10, ADAM17, MMP-13 or a combination thereof. In some embodiments, the metalloprotease is MMP-2. In some embodiments, the metalloprotease is MMP-2 or MMP-13. In some embodiments, the metalloprotease is MMP-2. In some embodiments, the metalloprotease is MMP-2. In some embodiments, the metalloprotease is MMP-2, MMP-13 or a combination thereof.

[0107] In some embodiments, the agent is selected from an antigen-binding fragment of an antibody, a Fab fragment, a single-chain antibody, a single-domain antibody, a small molecule, and a peptide that specifically binds to CD28. In some embodiments, the agent is a Fab fragment. In some embodiments, the agent is a single-chain antibody. In some embodiments, the agent is a single-domain antibody. In some embodiments, the agent is a peptide that specifically binds to CD28.

[0108] In some embodiments, the reagent lacks an Fc domain. In some embodiments, the reagent is an antigen binding domain lacking an Fc domain. In some embodiments, the reagent is a camelid antibody, a shark antibody, or a nanobody. In some embodiments, an antibody or fragment is fused to another protein or a fragment of a protein. In some embodiments, the second protein or fragment increases half-life, particularly in serum. In some embodiments, the half-life extension protein is human serum albumin. In some embodiments, the reagent is modified by producing a modified chemical substance that enhances half-life. In some embodiments, the modification is PEGylation and the chemical substance is polyethylene glycol. Those skilled in the art will recognize that any half-life extension protein or chemical agent, or modification known in the art, can be used.

[0109] Examples of agents include, but are not limited to, antibodies, antigen-binding fragments of antibodies, nanobodies, single-chain antibodies, single-domain antibodies, small molecules, peptides, and DARPins. In some embodiments, agents are selected from antibodies, antigen-binding fragments of antibodies, Fab fragments, nanobodies, single-chain antibodies, single-domain antibodies, small molecules, peptides, and DARPins. In some embodiments, agents are selected from antibodies, antigen-binding fragments of antibodies, Fab fragments, single-chain antibodies, single-domain antibodies, small molecules, and peptides that specifically bind to CD28. In some embodiments, the agent is a single-domain antibody. In some embodiments, the agent is a nanobody. In some embodiments, the agent is a VHH antibody. As used herein, the terms "single-domain antibody," "nanobody," and "VHH antibody" are synonymous and can be used interchangeably. In some embodiments, the peptide has specificity for binding to CD28. In some embodiments, the agent is a peptide that specifically binds to CD28. In some embodiments, the peptide is selected from antibodies, antigen-binding fragments of antibodies, Fab fragments, single-chain antibodies, single-domain antibodies, nanobodies, VHH antibodies, and antibody mimetics. As used herein, the term "antibody mimics" refers to an organic compound that can specifically bind to a target antigen. In some embodiments, antibody mimics are not structurally related to antibodies. Examples of antibody mimics include, but are not limited to, affilins, affimers, affitins, alpha antibodies (alphabodies), anticalins, avimers, DARPins, fynomers, Kunitz domain peptides, monobodies, and nano-CLAMPS. In some embodiments, the antibody mimic is a DARPin. All of these agents are known in the art and are known to block the interaction between a receptor and its ligand. Small molecules and proteins that can bind to mCD28 can block the cleavage site or may cause access to a hidden or impaired protease. In some embodiments, the protein is an antibody mimic. As used herein, the term "DARPin" refers to a designed ankyrin repeat protein. DARPins are typically engineered antibody mimics proteins that are typically highly specific for their protein targets. Therefore, the DARPin of CD28 can be an example of a reagent.

[0110] In some embodiments, the Fab fragment comprises a size of about 50 kDa. In some embodiments, the Fab fragment comprises a size of less than 100 kDa. In some embodiments, the Fab fragment comprises a size of less than 80 kDa. In some embodiments, the Fab fragment comprises a size of less than 70 kDa. In some embodiments, the Fab fragment comprises a size of less than 50 kDa. In some embodiments, the Fab fragment comprises a size of 50 kDa or less. In some embodiments, the single-chain antibody comprises a size of about 25 kDa. In some embodiments, the single-chain antibody comprises a size of less than 50 kDa. In some embodiments, the single-chain antibody comprises a size of less than 40 kDa. In some embodiments, the single-chain antibody comprises a size of less than 30 kDa. In some embodiments, the single-chain antibody comprises a size of less than 25 kDa. In some embodiments, the single-chain antibody comprises a size of 25 kDa or less. In some embodiments, the single domain antibody comprises a size of about 15 kDa. In some embodiments, the single domain antibody comprises a size between 10-17 kDa. In some embodiments, the single domain antibody comprises a size between 10-16 kDa. In some embodiments, the single domain antibody comprises a size between 10-15 kDa. In some embodiments, the single domain antibody comprises a size between 12-15 kDa. In some embodiments, the single domain antibody comprises a size between 12-16 kDa. In some embodiments, the single domain antibody comprises a size between 12-17 kDa. In some embodiments, the single domain antibody comprises a size less than 25 kDa. In some embodiments, the single domain antibody comprises a size less than 20 kDa. In some embodiments, the single domain antibody comprises a size less than 15 kDa. In some embodiments, the single domain antibody comprises a size of 15 kDa or less. Due to its small size, and having only 3 CDRs, the single domain antibody comprises a convex shape and binds its epitope only from one side. In contrast, Fab fragments and single chain antibodies contain 6 CDRs and bind epitopes from at least 2 sides. In some embodiments, binding to only 3 CDRs allows better access to the mCD28 stem region compared to binding to 6 CDRs. In some embodiments, the geometry of single domain antibody binding is better for access to the mCD28 stem region.

[0111] As used herein, the term "antibody" refers to a polypeptide or a group of polypeptides comprising at least one binding domain, which is formed by folding a polypeptide chain with a three-dimensional binding space having an inner surface shape and charge distribution that are complementary to the characteristics of an antigenic determinant. Antibodies usually have a tetrameric form, comprising two pairs of identical polypeptide chains, each pair having a "light" chain and a "heavy" chain. The variable region of each pair of light / heavy chains forms an antibody binding site. Antibodies can be oligoclonal, polyclonal, monoclonal, chimeric, camelidized, CDR-grafted, multispecific, bispecific, catalytic, humanized, fully human, anti-idiotypic, and antibodies that can be labeled in a soluble or bound form, as well as fragments (including epitope binding fragments), variants or derivatives thereof, alone or in combination with other amino acid sequences. Antibodies can be from any species. The term antibody also includes binding fragments, including, but not limited to, Fv, Fab, Fab', F(ab')2 single-chain antibodies (scFv), dimeric variable regions (Diabody) and disulfide bond variable regions (dsFv). Specifically, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing antigen binding sites. Antibody fragments may or may not be fused to another immunoglobulin domain, including but not limited to, an Fc region or a fragment thereof. Those skilled in the art will further recognize that other fusion products may be produced, including but not limited to scFv-Fc fusions, variable regions (e.g., VL and VH)-Fc fusions, and scFv-scFv-Fc fusions.

[0112] Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass.

[0113] The basic unit of naturally occurring antibody structure is a heterotetrameric glycoprotein complex of about 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains, linked together by non-covalent association and by disulfide bonds. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. There are five human antibody classes (IgG, IgA, IgM, IgD and IgE), and within these classes, various subclasses are identified based on structural differences, such as the number of immunoglobulin units in a single antibody molecule, the disulfide bond structure of the individual units, and the differences in chain length and sequence. The class and subclass of an antibody are its isotype. In some embodiments, the Fab fragment has a size less than 100, 90, 80, 75, 70, 65, 60, 55 or 50 kDa. Each possibility represents a separate embodiment of the present invention. In some embodiments, the Fab fragment has a size less than 50 kDa.

[0114] The amino terminal regions of heavy and light chains are more diverse in sequence than the carboxyl terminal regions, and are therefore referred to as variable domains. This part of the antibody structure confers the antigen binding specificity of the antibody. The heavy variable (VH) domain and the light variable (VL) domain together form a single antigen binding site, and therefore, the basic immunoglobulin unit has two antigen binding sites. Specific amino acid residues are thought to form an interface between the light chain and heavy chain variable domains (Chothia et al., J. Mol. Biol. 186, 651-63 (1985); Novotny and Haber, (1985) Proc. Natl. Acad. Sci. USA 824592-4596).

[0115] The carboxyl terminal parts of the heavy and light chains form constant domains, i.e., CH1, CH2, CH3, CL. Although there is less diversity in these domains, there are differences between animal species, and further, within the same individual, there are several different isotypes of antibodies, each with different functions.

[0116] The term "framework region" or "FR" refers to the amino acid residues in the variable domain of an antibody that are different from the hypervariable region amino acid residues defined herein. The term "hypervariable region" as used herein refers to the amino acid residues in the variable domain of an antibody that are responsible for antigen binding. The hypervariable region includes amino acid residues from the "complementarity determining region" or "CDR". The CDR is primarily responsible for binding to the epitope of the antigen. The scope of FR and CDR has been precisely defined (see, Kabat et al.).

[0117] Immunoglobulin variable domains can also be identified using the IMGT information system (www: / / imgt.cines.fr / ) ( / V-Quest) analysis to identify variable segments, including CDRs. See, e.g., Brochet, X. et al, Nucl. Acids Res. J6: W503-508 (2008).

[0118] Chothia et al. also defined a numbering system applicable to the variable domain sequences of any antibody. Those skilled in the art can clearly assign this "Chothia numbering" system to any variable domain sequence without relying on any experimental data other than the sequence itself. As used herein, "Chothia numbering" refers to the numbering system proposed by Chothia et al. Journal of Molecular Biology, "Canonical Structures for the Hypervariable regions of immunoglobulins" (1987) and Chothia's Nature, "Conformations of Immunoglobulin Hypervariable Regions" (1989).

[0119] As used herein, the terms "single-chain antibody" and "single-chain variable fragment" are used synonymously, and refer to a fusion protein of the variable region of the heavy chain and light chain of an immunoglobulin connected by a short peptide linker. In some embodiments, the single-chain antibody has a size less than 50, 45, 40, 35, 30, 25 or 20 kDa. Each possibility represents a separate embodiment of the present invention. In some embodiments, the single-chain antibody has a size less than 25 kDa. In some embodiments, the joint of the single-chain antibody is between 10 and 25 amino acids. In some embodiments, the joint is between 1-40, 5-40, 10-40, 1-35, 5-35, 10-35, 1-30, 5-30, 10-30, 1-25, 5-25 or 10-25 amino acids. Each possibility represents a separate embodiment of the present invention. In some embodiments, the single-chain antibody includes the heavy chain of antibody M9. In some embodiments, the single-chain antibody includes the light chain of antibody M9. In some embodiments, the single chain antibody comprises the CDRs of antibody M9.

[0120] As used herein, the terms "single domain antibody", "nanobody" and "VHH" are used synonymously and refer to antibody fragments consisting of a single monomeric variable antibody domain. In some embodiments, the single domain antibody is a camelid antibody. In some embodiments, the camelid is a camel, alpaca or llama. In some embodiments, the camelid is a camel. In some embodiments, the camelid is an alpaca. In some embodiments, the camelid is a llama. In some embodiments, the single domain antibody is a shark antibody.

[0121] Also, it has been indicated herein that the amino acid residues of Nanobodies are numbered according to the general VH numbering given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, Md., Publication No. 91), as applied to VHH domains of camelids in the article by Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun. 23; 240(1-2): 185-195; or as referred to herein. According to this numbering, FR1 of a Nanobody comprises the amino acid residues at positions 1-30, CDR1 of a Nanobody comprises the amino acid residues at positions 31-35, FR2 of a Nanobody comprises the amino acids at positions 36-49, CDR2 of a Nanobody comprises the amino acid residues at positions 50-65, FR3 of a Nanobody comprises the amino acid residues at positions 66-94, CDR3 of a Nanobody comprises the amino acid residues at positions 95-102, and FR4 of a Nanobody comprises the amino acid residues at positions 103-113. In this regard, it should be noted that - as is well known in the art for VH domains and VHH domains - the total number of amino acid residues in each CDR may be different and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by the Kabat numbering). This means that, in general, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence. In general, however, it can be said that, according to the numbering of Kabat, and irrespective of the number of amino acid residues of the CDR, position 1 according to the Kabat numbering corresponds to the start of FR1 and vice versa, position 36 according to the Kabat numbering corresponds to the start of FR2 and vice versa, position 66 according to the Kabat numbering corresponds to the start of FR3 and vice versa, and position 103 according to the Kabat numbering corresponds to the start of FR4 and vice versa.

[0122] An alternative method for numbering the amino acid residues of a VH domain (which can also be applied in an analogous manner to VHH domains from camelid antibodies and nanobodies) is the method described by Chothia et al. (Nature 342, 877-883 (1989)), the so-called "AbM definition" and the so-called "contact definition". However, in the present specification, aspects and figures, unless otherwise indicated, the numbering according to Kabat by Riechmann and Muyldermans for VHH domains will be followed.

[0123] As used herein, the term "humanized antibody" refers to an antibody from a non-human species whose protein sequence has been modified to increase similarity to a human antibody. Humanized antibodies can be produced by recombinant DNA that produces CDRs of non-human antibodies that are surrounded by sequences similar to human antibodies. In some embodiments, humanized antibodies are chimeric antibodies. In some embodiments, humanization includes inserting the CDRs of the present invention into a human antibody scaffold or skeleton. Humanized antibodies are well known in the art, and any method for producing humanized antibodies that retain the CDRs of the present invention can be used.

[0124] As used herein, the term "monoclonal antibody" or "mAb" refers to an antibody obtained from a substantially homotypic antibody population, that is, the individual antibodies constituting the population are identical and / or bind to the same epitope, except for variants that may occur during the production of the monoclonal antibody, which are usually present in small amounts. Contrary to polyclonal antibody preparations that typically include different antibodies for different determinants (epitopes), each monoclonal antibody is directed to a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are not contaminated by other immunoglobulins. The modifier "monoclonal" represents the characteristics of the antibody obtained from a substantially homotypic antibody population, and should not be construed as being produced by any specific preparation method. The monoclonal antibody used according to the method provided herein can be prepared by the hybridoma method first described by Kohler et al. (Nature 256:495 (1975)), or can be prepared by a recombinant DNA method (see, e.g., U.S. Patent No. 4,816,567). For example, "monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described by Clackson et al. (Nature 352:624-628 (1991)) and Marks et al. (J. Mol. Biol. 222:581-597 (1991)).

[0125] The mAb of the present invention can be any immunoglobulin class, including IgG, IgM, IgD, IgE or IgA. The hybridoma producing mAb can be cultured in vitro or in vivo. High titer mAb can be obtained in vivo production, wherein cells from a single hybridoma are injected intraperitoneally into pristine-primed Balb / c mice to produce ascites containing high concentrations of the desired mAb. mAbs of isotype IgM or IgG can be purified from these ascites fluids or culture supernatants using column chromatography well known to those skilled in the art.

[0126] "Antibody fragments" include a portion of an intact antibody, preferably including its antigen binding region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; tandem diabodies (taDb), linear antibodies (e.g., U.S. Pat. No. 5,641,870, Example 2; Zapata et al. Protein Eng. 8(10): 1057-1062 (1995)); one-armed antibodies, single variable domain antibodies, minibodies, single-chain antibody molecules; multispecific antibodies formed by antibody fragments (e.g., including but not limited to, Db-Fc, taDb-Fc, taDb-CH3, (scFv)4-Fc, di-scFv, bi-scFv, or tandem (di,tri)-scFv); and bispecific T cell engagers (BiTEs).

[0127] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment produces the F(ab')2 fragment, which has two antigen-binding sites and is still capable of cross-linking antigen.

[0128] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and antigen binding site. This region consists of a dimer of a heavy chain and a light chain variable domain in tight, non-covalent association. The three surfaces of the VH-VL dimer are in this configuration. In general, the six hypervariable regions give the antibody antigen binding specificity. However, even a single variable domain (or half of an Fv that includes only three hypervariable regions specific for an antigen) has the ability to recognize and bind to an antigen, although the affinity is lower than that of the entire binding site.

[0129] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that a few residues are added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation for Fab' herein, in which the cysteine ​​residue(s) of the constant domains carry at least one free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0130] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.

[0131] According to the amino acid sequence of the constant domain of its heavy chain, antibodies can be assigned to different classes. There are five main classes of complete antibodies: IgA, IgD, IgE, IgG and IgM, several of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The heavy chain constant domains corresponding to different classes of antibodies are respectively referred to as α, δ, ε, γ and μ. The subunit structure and three-dimensional configuration of different classes of immunoglobulins are well known.

[0132] "Single-chain Fv" or "scFv" antibody fragments include the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further includes a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0133] The term "diabody" refers to a small antibody fragment with two antigen binding sites, which includes a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to pair between the two domains on the same chain, these domains are forced to pair with the complementary domains of another chain and produce two antigen binding sites. Diabody production is known in the art and is described in Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

[0134] The term "multispecific antibody" is used in the broadest sense and specifically encompasses antibodies with polyepitopic specificity. Such multispecific antibodies include, but are not limited to, antibodies comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) wherein the VHVL unit has polyepitopic specificity, antibodies having two or more VL and VH domains wherein each VHVL unit binds to a different epitope, antibodies having two or more single variable domains wherein each single variable domain binds to a different epitope, full length antibodies, antibody fragments (such as Fab, Fv, dsFv, scFv), diabodies, bispecific diabodies, triabodies, trifunctional antibodies, covalently or non-covalently linked antibody fragments. "Polyepitopic specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different targets (one or more).

[0135] Monoclonal antibodies can be prepared using methods known in the art. Examples include various techniques, such as those of Kohler, G. and Milstein, C, Nature 256:495-497 (1975); Kozbor et al, Immunology Today 4:72 (1983); Cole et al, pg.77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

[0136] In addition to conventional methods for producing antibodies in vivo, phage display technology can also be used to produce antibodies in vitro. Compared with conventional antibody production, the production of such recombinant antibodies is much faster, and recombinant antibodies can be produced for a large number of antigens. In addition, when using conventional methods, many antigens have been shown to be non-immunogenic or highly toxic and therefore cannot be used to produce antibodies in animals. In addition, the affinity maturation (i.e., increasing affinity and specificity) of recombinant antibodies is very simple and relatively fast. Finally, a large number of different antibodies against a specific antigen can be produced in one selection procedure. In order to produce recombinant monoclonal antibodies, people can use various methods based on display libraries to produce a large number of antibodies with different antigen recognition sites. Such libraries can be constructed in a variety of ways: synthetic libraries can be generated by cloning synthetic CDR3 regions in heavy chain germline gene libraries, thereby generating large antibody libraries, from which recombinant antibody fragments with various specificities can be selected. People can use human lymphocyte libraries as starting materials for constructing antibody libraries. A primitive library of human IgM antibodies can be constructed, thereby creating a human library with a wide variety of diversity. This method has been widely and successfully used to select a large number of antibodies against different antigens. Protocols for phage library construction and recombinant antibody selection are provided in the well-known reference text Current Protocols in Immunology, Colligan et al (Eds.), John Wiley & Sons, Inc. (1992-2000), Chapter 17, Section 17.1.

[0137] Non-human antibodies can be humanized by any method known in the art. In one method, non-human complementary determining regions (CDRs) are inserted into human antibodies or consensus antibody framework sequences. Further changes can then be introduced into the anti-antibody framework to adjust affinity or immunogenicity.

[0138] In some embodiments, antibodies and parts thereof include: antibodies, antibody fragments, Fab and F(ab')2, single domain antigen binding recombinant fragments and natural nanobodies. In some embodiments, the antigen binding fragment is selected from Fv, Fab, F(ab')2, scFV or scFV2 ​​fragments.

[0139] In some embodiments, the invention provides nucleic acid sequences encoding the antibodies or antigen-binding portions of the invention.

[0140] For example, a polynucleotide can encode a complete immunoglobulin molecule chain, such as a light chain or a heavy chain. A complete heavy chain includes not only a heavy chain variable region (VH) but also a heavy chain constant region (CH), which generally includes three constant domains: CH1, CH2 and CH3; and a "hinge" region. In some cases, the presence of a constant region is desirable.

[0141] Other polypeptides that can be encoded by polynucleotides include antigen-binding antibody fragments, such as single domain antibodies ("dAbs"), Fv, scFv, Fab' and CHI, and CK or CL domains have been removed. Because mini antibodies are smaller than conventional antibodies, all of them should achieve better tissue penetration in clinical / diagnostic use, but they should retain higher binding affinity than monovalent antibody fragments such as dAbs, which are bivalent. Therefore, unless the context otherwise provides, the term "antibody" used herein includes not only whole antibody molecules, but also antigen-binding antibody fragments of the types discussed above. Each framework region present in the encoded polypeptide may include at least one amino acid substitution relative to the corresponding human receptor framework. Therefore, for example, the framework region may include a total of three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions relative to the receptor framework region. In view of the characteristics of the individual amino acids comprising the disclosed protein product, those skilled in the art will recognize some reasonable substitutions. Amino acid substitutions, ie, "conservative substitutions," may be made, for example, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved.

[0142] Suitably, the polynucleotides described herein may be isolated and / or purified.In some embodiments, the polynucleotide is an isolated polynucleotide.

[0143] As used herein, the term "non-naturally occurring" substances, compositions, entities, and / or any combination of substances, compositions or entities, or any grammatical variations thereof, is a conditional term that expressly excludes, but only excludes, those forms of substances, compositions, entities, and / or combinations of substances, compositions, entities that are known to one of ordinary skill in the art as "naturally occurring" or that may at any time be determined or interpreted by a judge, administrative or judicial body to be "naturally occurring."

[0144] On the other hand, an agent is provided, which includes three CDRs, wherein CDR1 includes the amino acid sequence described in SEQ ID NO:33 (INAMG), CDR2 includes the amino acid sequence described in SEQ ID NO:34 (AISGGGDTYYADSVKG), and CDR3 includes the amino acid sequence described in SEQ ID NO:35 (DLYGSDYWD).

[0145] By way of another aspect, an agent is provided which comprises three CDRs, wherein CDR1 comprises the amino acid sequence recited in SEQ ID NO:36 (INAMA), CDR2 comprises the amino acid sequence recited in SEQ ID NO:37 (AITSSGSTNYANSVKG), and CDR3 comprises the amino acid sequence recited in SEQ ID NO:38 (DEYGSDYWI).

[0146] By way of another aspect, an agent is provided which comprises three CDRs, wherein CDR1 comprises the amino acid sequence recited in SEQ ID NO:33 (INAMG), CDR2 comprises the amino acid sequence recited in SEQ ID NO:39 (AITSGGSTNYADSVKG), and CDR3 comprises the amino acid sequence recited in SEQ ID NO:40 (DLYGEDYWI).

[0147] In some embodiments, the CDRs are numbered according to the Abm numbering method. In some embodiments, the CDRs are numbered according to the Chothia numbering method. In some embodiments, the CDRs are numbered according to the Kabat numbering method.

[0148] In some embodiments, CDR1 comprises the amino acid sequence set forth in SEQ ID NO:41 (INAMX1), wherein X1 is G or A. In some embodiments, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:42 (AIX1X2X3GX4TX5YAX6SVKG), wherein X1 is S or T, X2 is G or S, X3 is G or S, X4 is D or S, X5 is Y or N, and X6 is D or N. In some embodiments, CDR3 comprises the amino acid sequence set forth in SEQ ID NO:43 (DX1YGX2DYWX3), wherein X1 is E or L, X2 is E or S, and X3 is D or I. In some embodiments, CDR3 comprises the amino acid sequence set forth in SEQ ID NO:44 (DX1YGSDYWX2), wherein X1 is E or L, and X2 is D or I.

[0149] In some embodiments, the agent is a single domain antibody. In some embodiments, the agent is a VHH antibody. In some embodiments, the agent is a camelid antibody. In some embodiments, the camelid is a llama. In some embodiments, the agent does not include other CDRs in addition to the CDRs described above.

[0150] In some embodiments, an agent comprises a sequence comprising and / or consisting of EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVTVSS (SEQ ID NO: 30).

[0151] In some embodiments, an agent comprises a sequence comprising and / or consisting of EVQLVESGGGLVQAGGSLRLSCAASGSLFSINAMAWYRQAPGKQRELVAAITSSGSTNYANSVKGRFTVSRDNAKNTMYLQMNSLKPEDTAVYYCVVDEYGSDYWIWGQGTQVTVSS (SEQ ID NO: 31).

[0152] In some embodiments, an agent comprises a sequence comprising and / or consisting of QVQLVESGGGLVQAGGSLRLSCAASGSIFSINAMGWYRQAPGKQRERVAAITSGGSTNYADSVKGRFTISRDNAKNTVYLQMNNLEPRDAGVYYCVVDLYGEDYWIWGQGTQVTVSS (SEQ ID NO: 32).

[0153] In some embodiments, the VHH sequence further includes a His tag. In some embodiments, the His tag is at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 histidine residues. Each possibility represents a separate embodiment of the present invention. In some embodiments, the His tag consists of 6 histidine residues. In some embodiments, the His tag is connected to the VHH through a joint. In some embodiments, the joint is a peptide joint. In some embodiments, the joint is an alanine repeat joint. In some embodiments, the alanine repeat comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 alanine residues. Each possibility represents a separate embodiment of the present invention. In some embodiments, the alanine repeat joint consists of 3 alanine residues. In some embodiments, the His-tag is six His tags.

[0154] In some embodiments, the VHH sequences found to specifically bind to the stem region of human CD28 and include a His tag are: EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVTVSSAAAHHHHHH (SEQ ID NO:45, clone 2A1); EVQLVESGGGLVQAGGSLRLSCAASGSLFSINAMAWYRQAPGKQRELVAAITSSGSTNYANSVKGRFTVSRDNAKNTMYLQMNSLKPEDTAVYYCVVDEYGSDYWIWGQGTQVTVSSAAAHHHHHH (SEQ ID NO:45, clone 2A1); NO:46, clone 4A4); and QVQLVESGGGLVQAGGSLRLSCAASGSIFSINAMGWYRQAPGKQRERVAAITSGGSTNYADSVKGRFTISRDNAKNTVYLQMNNLEPRDAGVYYCVVDLYGEDYWIWGQGTQVTVSSAAAHHHHHH (SEQ ID NO:47, clone 4A1).

[0155] By another aspect, an agent is provided, which includes three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein: CDR-H1 includes the amino acid sequence recited in SEQ ID NO: 11 (GYTLTNY), CDR-H2 includes the amino acid sequence recited in SEQ ID NO: 12 (NTYTGK), CDR-H3 includes the amino acid sequence recited in SEQ ID NO: 13 (GDANQQFAY), CDR-L1 includes the amino acid sequence recited in SEQ ID NO: 14 (KASQDINSYLS), CDR-L2 includes the amino acid sequence recited in SEQ ID NO: 15 (RANRLVD), and CDR-L3 includes the amino acid sequence recited in SEQ ID NO: 16 (LQYDEFPPT). The antibody is referred to herein as M9.

[0156] In some embodiments, the agent includes three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein: CDR-H1 includes the amino acid sequence described in SEQ ID NO:17 (GFTFSSYYMS), CDR-H2 includes the amino acid sequence described in SEQ ID NO:18 (TISDGGDNTYYAGTVTG), CDR-H3 includes the amino acid sequence described in SEQ ID NO:19 (IHWPYYFDS), CDR-L1 includes the amino acid sequence described in SEQ ID NO:20 (RASSSVSYMN), CDR-L2 includes the amino acid sequence described in SEQ ID NO:21 (ATSDLAS), and CDR-L3 includes the amino acid sequence described in SEQ ID NO:22 (QQWSSHPPT).

[0157] In some embodiments, the agent comprises a heavy chain comprising the following amino acid sequence: DVKLVESGGGLVKLGGSLKLSCVASGFTFSSYYMSWVRQTPEKRLEWVATISDGGDNTYYAGTVTGRFTISRDFAKNTLYLQMNSLTSEDTAVYYCARIHWPYYFDSWGQGTTLTVSS (SEQ ID NO: 23). In some embodiments, the variable region of the heavy chain comprises and / or consists of SEQ ID NO: 23. In some embodiments, the agent includes a heavy chain comprising a polypeptide encoded by the following nucleic acid sequence: GACGTGAAGCTCGTGGAGTCTGGGGGAGGCTTAGTGAAGCTTGGAGGGTCCCTGAAACTCTCCTGTGTAGCCTCTGGATTCACTTTCAGTAGCTATTACATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCGACCATAAGTGATGGTGGTGATAACACCTACTACGCAGGCACTGTGACGGGCCGATTCACCATCTCCAGAGACTTTGCCAAGAACACCCTGTACCTGCAAATGAACAGTCTGACCTCTGAGGACACAGCCGTGTATTACTGTGCAAGAATTCATTGGCCTTACTATTTTGACTCCTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO: 24). In some embodiments, the heavy chain consists of SEQ ID NO: 24. Antibody M9 was sequenced and found to have a heavy chain consisting of SEQ ID NO: 24. The CDRs of the heavy chain as determined using the Chothia protocol are SEQ ID NOs: 17-19.

[0158] In some embodiments, the agent comprises a light chain comprising the following amino acid sequence: QFVLSQSPAILSASPGEMLTMTCRASSSVSYMNWYQQKPGSSPKPWIYATSDLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSHPPTFGGGTKLEIR (SEQ ID NO: 25). In some embodiments, the variable region of the light chain comprises or consists of SEQ ID NO: 25. In some embodiments, the agent includes a light chain comprising a polypeptide encoded by the following nucleic acid sequence: CAATTTGTTCTCTCCCAGTCTCCAGCAATCCTGTCTGCATCTCCCGGGGAGATGCTCACAATGACTTGCAGGGCCAGCTCAAGTGTAAGTTATATGAACTGGTATCAGCAGAAGCCAGGATCTTCCCCCAAACCCTGGATTTATGCCACATCCGACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTATTCTCTCACAATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTCACCCACCCACGTTCGGAGGGGGGACCAAGCTGGAAATAAGA (SEQ ID NO: 26). In some embodiments, the light chain consists of SEQ ID NO: 26. Antibody M9 was sequenced and found to have a light chain consisting of SEQ ID NO: 26. The CDRs of the light chain as determined using the Chothia protocol are SEQ ID NOs: 20-22.

[0159] In some embodiments, the agent binds as a monomer. In some embodiments, the agent binds as a dimer. In some embodiments, the agent binds as a monomer and / or dimer. In some embodiments, the agent binds as a dimer, but does not crosslink and / or activate mCD28. In some embodiments, the agent binds as a dimer, but only binds to a single molecule of CD28. In some embodiments, the agent binds to monomeric CD28. In some embodiments, the agent binds to dimeric CD28. In some embodiments, the agent binds to monomeric and / or dimeric CD28.

[0160] In some embodiments, the agent is not a CD28 agonist. In some embodiments, the agent is not a CD28 antagonist. In some embodiments, the agent is neither a CD28 agonist nor an antagonist.

[0161] The term "agonist" generally refers to a molecule, compound or reagent that binds to a receptor and fully or partially activates a receptor. In some embodiments, an agonist binds at the same site as a natural ligand. In some embodiments, an agonist binds at an allosteric site that is different from the binding site of a natural ligand. The term "antagonist" generally refers to a molecule, compound or reagent that binds to a receptor at the same site or another site as an agonist, does not activate the receptor, and performs one or more of the following operations: interferes with or blocks the activation of a natural ligand to a receptor and interferes with or blocks the activation of a receptor agonist to a receptor. In some embodiments, the antibodies of the present invention bind to mCD28 but do not activate or block the activation of a receptor. In some embodiments, they do not block the activation by CD86. In some embodiments, the antibodies of the present invention do not bind to mCD28.

[0162] As used herein, "direct agonist / antagonist" refers to a molecule that binds to a receptor (mCD28) and increases / reduces the signaling of the molecule by binding. In the case of mCD28, an agonist will bind to mCD28 and increase mCD28 signaling in cells by binding. In some embodiments, an agonist increases T cell activation. In some embodiments, an agonist increases T cell proliferation. In some embodiments, an agonist increases proinflammatory cytokine secretion. Proinflammatory cytokines are well known in the art and are known to be secreted by activated T cells. Examples of proinflammatory cytokines include, but are not limited to, TNFα, IFNγ, IL-1B, IL-2, and IL-6. In some embodiments, the proinflammatory cytokine is IFNγ. In some embodiments, the proinflammatory cytokine is IL-2. In the case of mCD28, an antagonist will bind to mCD28 and reduce mCD28 signaling in cells by binding. In some embodiments, an antagonist reduces T cell activation, reduces T cell proliferation, and / or reduces proinflammatory cytokine secretion. Molecules that affect receptor signaling by contacting its ligand, contacting an inhibitor, contacting a co-receptor, or contacting any molecule other than the receptor in question to alter receptor signaling are not considered direct agonists / antagonists. In some embodiments, the agents of the invention contact sCD28 in serum and thereby allow for increased signaling through mCD28 on cells. Although the result is an increase in mCD28 signaling, the antibody is not an mCD28 agonist or direct agonist because its binding to mCD28 does not increase receptor signaling.

[0163] In some embodiments, the agent does not bind to the ligand binding domain of mCD28. In some embodiments, the agent does not conceal or block access to the ligand binding domain. In some embodiments, the agent does not bind, conceal or block access to the IgV domain of sCD28. In some embodiments, the IgV domain is a ligand binding domain. In some embodiments, the ligand binding domain comprises amino acids 28-137 of SEQ ID NO: 1. In some embodiments, the ligand binding domain comprises or consists of the following amino acid sequence: MLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKG (SEQ ID NO: 8). In some embodiments, the agent does not inhibit the binding of sCD28 to the ligand. In some embodiments, the CD28 ligand is selected from: CD80, CD86 and ICOSL. In some embodiments, the CD28 ligand is CD86. In some embodiments, the CD28 ligand is CD80. In some embodiments, the CD28 ligand is ICOSL. In some embodiments, CD86 is CD86-Fc. In some embodiments, CD80 is CD80-Fc.

[0164] In some embodiments, the agent binds to the stem region of CD28. In some embodiments, the agent binds to the membrane proximal region of mCD28. In some embodiments, the stem region comprises the sequence GKHLCPSPLFPGPSKP (SEQ ID NO: 9). In some embodiments, the stem region comprises the sequence KGKHLCPSPLFPGPS (SEQ ID NO: 27). In some embodiments, the stem region comprises or consists of the sequence HVKGKHLCPSPLFPGPSKP (SEQ ID NO: 10). In some embodiments, the agent binds to monomeric sCD28. In some embodiments, the agent binds to dimeric sCD28. In some embodiments, the agent binds to monomeric sCD28, dimeric sCD28, or both. In some embodiments, the agent binds to monomeric CD28 but does not bind to dimeric CD28. In some embodiments, a fragment of the extracellular domain of CD28 is the stem region. In some embodiments, the agent that binds to CD28 prevents the shearing of CD28. In some embodiments, the agent that binds to CD28 prevents CD28 from falling off the cell.

[0165] In some embodiments, the agent binds at a cleavage site in the stem region. In some embodiments, the agent binds at a cleavage site within mCD28. In some embodiments, the agent binds at a cleavage site of at least one protease. In some embodiments, the agent binds at a cleavage site of MMP-2.

[0166] In some embodiments, the agent does not bind to the ligand binding domain of mCD28. In some embodiments, the agent does not conceal or block access to the ligand binding domain. In some embodiments, the agent binds at the cleavage site. In some embodiments, the agent conceals, blocks or blocks access to the cleavage site. In some embodiments, the agent binds, blocks, blocks or conceals the protease cleavage site. In some embodiments, the agent does not bind to the protease cleavage site but blocks the site. In some embodiments, the agent blocks access to the protease cleavage site. In some embodiments, the agent produces a steric hindrance that blocks the protease cleavage site. In some embodiments, the agent does not bind to the protease cleavage site, but the binding of the agent produces a conformational change in mCD28 that blocks the protease cleavage site. In some embodiments, the binding of the agent produces a conformational change in mCD28 that blocks the protease cleavage site. In some embodiments, the protease is MMP-2. In some embodiments, the protease is MMP-13. In some embodiments, the cleavage site is a cleavage motif. In some embodiments, the MMP-2 cleavage motif is PXX / X, where the last X is a hydrophobic residue. In some embodiments, the PXX / X motif in CD28 is PSP / L. In some embodiments, the protease cleavage site is amino acids 142-145 (PSPL) of SEQ ID NO: 1. In some embodiments, the protease cleavage site is amino acids 127-130 (PSPL) of SEQ ID NO: 2. In some embodiments, the protease cleavage site is amino acids 9-12 (PSPL) of SEQ ID NO: 10. In some embodiments, the agent blocks access of the protease to the cleavage site. In some embodiments, the agent binds to the PSPL in the stem domain of mCD28.

[0167] In some embodiments, the cleavage site is before leucine. In some embodiments, the cleavage site is before valine. In some embodiments, the cleavage site is before an aromatic amino acid. In some embodiments, the cleavage site is before leucine, valine and / or an aromatic amino acid. In some embodiments, the aromatic amino acid is selected from phenylalanine, tryptophan, tyrosine and histidine. In some embodiments, the cleavage site is before any one of histidine 134, valine 135, histidine 139, leucine 140, leucine 145 and phenylalanine 146 of SEQ ID NO:1. In some embodiments, the cleavage site is before histidine 134, valine 135, histidine 139, leucine 140, leucine 145, or phenylalanine 146 of SEQ ID NO:1. Each possibility represents a separate embodiment of the present invention. In some embodiments, the cleavage site is before leucine 145 of SEQ ID NO:1. In some embodiments, the cleavage site is before leucine 145 of SEQ ID NO:1. In some embodiments, the cleavage site is before leucine 127 of SEQ ID NO:2.

[0168] In some embodiments, the agent does not bind to the stem region of CD28 with a mutated cleavage site. In some embodiments, the stem region of CD28 with a mutated cleavage site is not a substrate for a protease. In some embodiments, the stem region of CD28 with a mutated cleavage site is not a substrate for a metalloprotease. In some embodiments, the stem region of CD28 with a mutated cleavage site is not a substrate for a matrix metalloprotease. In some embodiments, the stem region of CD28 with a mutated cleavage site is not a substrate for a matrix metalloprotease 2 (MMP-2). In some embodiments, the stem region of CD28 with a mutated cleavage site is not a substrate for a matrix metalloprotease 13 (MMP-13). In some embodiments, the mutated cleavage site is a mutation of Leucine 145 of SEQ ID NO: 1. In some embodiments, the mutated cleavage site is an amino acid substitution of Leucine 145 of SEQ ID NO: 1. In some embodiments, the amino acid substitution of Leucine 145 of SEQ ID NO: 1 is lysine.

[0169] In some embodiments, the agent does not regulate CD28 function and / or signal transduction. In some embodiments, the agent does not degrade mCD28. In some embodiments, the agent does not cause or promote mCD28 degradation. In some embodiments, signal transduction is mCD28-mediated immune cell activation. In some embodiments, the agent does not inhibit immune cell activation. In some embodiments, the agent does not induce CD28 receptor internalization or circulation. The co-stimulation of mCD28 is critical to the immune activation of T cells. Proteolytic cleavage removes the ligand binding domain in the extracellular region of CD28 from the transmembrane and cytoplasmic parts of the protein retained in the membrane. Therefore, the sheared CD28 cannot conduct signals and cannot contribute to T cell activation. Therefore, agents that block shearing and are also antagonists do not allow mCD28 activation. Similarly, agents that block shearing but are also agonists can induce abnormal T cell activation and potentially autoimmune reactions.

[0170] In some embodiments, the agent does not reduce the surface level of mCD28 on immune cells. In some embodiments, the immune cells are T cells. In some embodiments, the agent reduces the surface level of mCD28 by less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 7%, 5%, 3%, 2% or 1%. Each possibility represents a separate embodiment of the present invention.

[0171] In some embodiments, the combination of the reagent and the cell does not kill the cell. In some embodiments, the combination of the reagent and the cell does not cause the death of the cell. In some embodiments, the reagent does not induce antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the reagent does not induce complement-dependent cytotoxicity (CDC). In some embodiments, the reagent does not induce ADCC and / or CDC. In some embodiments, the reagent is an antibody and includes an IgG2 or IgG4 domain. In some embodiments, the antibody includes an IgG2 domain. In some embodiments, the antibody includes an IgG4 domain. In some embodiments, the antibody includes an IgG1 or IgG3, which is mutated to reduce cell death mediated by the combination of the antibody. In some embodiments, the mutation mutates the Fc receptor binding domain. In some embodiments, the Fc domain of the antibody is engineered or mutated to reduce CDC, ADCC or both. Fc engineering is well known in the art, and any mutation or amino acid change known to reduce antibody-mediated cell killing can be used.

[0172] In some embodiments, the agent lacks an Fc domain. In some embodiments, the agent is an antigen binding domain lacking an Fc domain. In some embodiments, the agent is a single domain antibody. In some embodiments, the agent is a camelid antibody, a shark antibody, or a nanobody.

[0173] In some embodiments, the agent is a non-antibody protein. In some embodiments, the agent is a small molecule. In some embodiments, the agent is a nucleic acid molecule. In some embodiments, the agent is a synthetic peptide. In some embodiments, the agent is a synthetic binding protein. In some embodiments, the synthetic peptide is based on a non-antibody scaffold. In some embodiments, the agent is an antibody mimetic. In some embodiments, the molar mass of the antibody mimetic is less than 100, 90, 80, 70, 60, 50, 40, 30 or 20 kDa. Each possibility represents a separate embodiment of the present invention. In some embodiments, the agent is a nucleic acid aptamer. In some embodiments, the aptamer is DNA. In some embodiments, the aptamer is RNA. In some embodiments, the aptamer is DNA or RNA. Examples of antibody mimetic include, but are not limited to, affilins, affimers, affitins, alpha antibodies, anticalins, avimers, DARPins, fynomers, Kunitz domain peptides, monoclonal antibodies, and nano-CLAMPS. In some embodiments, the antibody mimetic is a DARPin.

[0174] In some embodiments, the agent inhibits protease cleavage by at least one protease. In some embodiments, the protease is a metalloprotease. In some embodiments, the protease is a matrix metalloprotease. In some embodiments, the protease is a serine protease. In some embodiments, the protease is a cysteine ​​protease. In some embodiments, the protease is a threonine protease. In some embodiments, the protease is a serine, cysteine ​​or threonine protease. In some embodiments, the protease is an aspartic protease. In some embodiments, the protease is a glutamic protease. In some embodiments, the protease is selected from aspartic acid, glutamic acid, serine, cysteine ​​and threonine protease. In some embodiments, the protease is an asparagine peptide cleavage enzyme. In some embodiments, the protease is a shedding enzyme (sheddase). In some embodiments, the metalloprotease is an exopeptidase. In some embodiments, the metalloprotease is an endopeptidase. In some embodiments, the metalloprotease is an exopeptidase or an endopeptidase. In some embodiments, the metalloprotease is zinc catalyzed. In some embodiments, the metalloprotease is cobalt catalyzed. In some embodiments, the metalloprotease is matrix metalloprotease-2 (MMP-2). In some embodiments, the metalloproteinase is matrix metalloproteinase-13 (MMP-13). In some embodiments, the metalloproteinase is ADAM10. In some embodiments, the metalloproteinase is ADAM17. In some embodiments, the metalloproteinase is ADAM10, MMP-2, and / or ADAM17. In some embodiments, the metalloproteinase is ADAM10, MMP-2, MMP-13 and / or ADAM17. In some embodiments, the metalloproteinase is MMP-2, ADAM10, ADAM17, or a combination thereof. In some embodiments, the metalloproteinase is MMP-2, MMP-13, ADAM10, ADAM17, or a combination thereof.

[0175] How to use

[0176] By way of another aspect, provided is a method of treating and / or preventing cancer in a subject in need thereof, the method comprising administering an agent of the invention.

[0177] By way of another aspect, provided is a method of improving immunotherapy in a subject in need thereof, the method comprising administering an agent of the invention.

[0178] By way of another aspect, provided is a method of reducing sCD28 in a subject in need thereof, the method comprising administering an agent of the invention.

[0179] In some embodiments, immunotherapy is an immunotherapy based on PD-1 and / or PD-L1. In some embodiments, immunotherapy based on PD-1 / PD-L1 includes administering anti-PD1 or anti-PD-L1 antibodies. In some embodiments, therapy includes blocking of PD-1 restriction points. In some embodiments, immunotherapy includes administering allogeneic, isogenic, or autologous immune cells to the subject. In some embodiments, the immune cells are T cells. In some embodiments, the subject in need of immunotherapy has cancer. In some embodiments, the subject has cancer. In some embodiments, the cancer is sCD28 positive cancer. In some embodiments, the cancer is sCD28 high cancer. In some embodiments, the subject is at risk of developing cancer.

[0180] As used herein, the terms "treatment" and "treating" a disease, disorder, or condition include alleviation of at least one symptom thereof, reduction of its severity, or inhibition of its progression. Treatment does not necessarily mean that the disease, disorder, or condition is completely cured. To be an effective treatment, the compositions useful herein need only reduce the severity of the disease, disorder, or condition, reduce the severity of the symptoms associated therewith, or improve the quality of life of the patient or subject.

[0181] In some embodiments, reducing comprises administering at least one reagent of the present invention to the subject. As used herein, the term "administering" and similar terms refer to any method of delivering a composition containing an active agent to the subject in a reasonable medical practice in a manner that provides a therapeutic effect. One aspect of this theme provides oral administration of a therapeutically effective amount of the reagent of the present invention to a patient in need thereof. Other suitable routes of administration may include parenteral, subcutaneous, intravenous, intramuscular or intraperitoneal.

[0182] By another aspect, a pharmaceutical composition comprising an agent of the invention and a pharmaceutically acceptable carrier, adjuvant or excipient is provided. In some embodiments, administration is with a pharmaceutical composition of the invention.

[0183] As used herein, the term "carrier", "excipient" or "adjuvant" refers to any component of a pharmaceutical composition that is not an active agent. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, any type of formulation aid, or simply a sterile aqueous medium, such as saline. Some examples of materials that can serve as pharmaceutically acceptable carriers are sugars (such as lactose, glucose and sucrose), starches (such as corn starch and potato starch), cellulose and its derivatives (such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate); tragacanth powder; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil, etc.; polyols such as glycerol, sorbitol, mannitol, polyethylene glycol, etc.; esters such as ethyl oleate and ethyl laurate, agar; buffers such as magnesium hydroxide, aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethanol and phosphate buffer solutions, and other nontoxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of materials that can be used as carriers herein include sugar, starch, cellulose and its derivatives, powdered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffered solution, cocoa butter (suppository base), emulsifiers, and other non-toxic pharmaceutically compatible substances for other pharmaceutical preparations. Wetting agents and lubricants such as sodium lauryl sulfate, as well as colorants, flavoring agents, excipients, stabilizers, antioxidants, and preservatives may also be present. Any non-toxic, inert, and effective carrier can be used to formulate the compositions contemplated herein. Suitable pharmaceutically acceptable carriers, excipients and diluents in this regard are well known to those skilled in the art, such as those described in The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, NJ (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and "Inactive Ingredient Guide," US Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, the entire contents of which are hereby incorporated by reference in their entirety.Examples of pharmaceutically acceptable excipients, carriers and diluents that can be used in the present compositions include distilled water, saline, Ringer's solution, dextrose solution, Hank's solution and DMSO. These additional inactive ingredients as well as effective formulations and administration procedures are well known in the art and described in standard textbooks, such as Goodman and Gillman's: The Pharmacological Bases of Therapeutics, 8th Ed., Gilman et al. Eds. Pergamon Press (1990); Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005), each of which is incorporated herein by reference in its entirety. The compositions described herein may also be included in artificially generated structures, such as liposomes, ISCOMS, sustained-release particles and other carriers that increase the half-life of peptides or polypeptides in serum. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, etc. The liposomes used with the currently described peptides are formed by standard vesicle-forming lipids, which typically include neutral and negatively charged phospholipids and sterols, such as cholesterol. The choice of lipids is typically determined by considerations such as liposome size and stability in blood. A variety of methods can be used to prepare liposomes, such as Coligan, JE et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, and also see U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028 and 5,019,369.

[0184] Carriers may comprise in total from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0185] In some embodiments, the methods of the present invention do not degrade or cause degradation of mCD28. In some embodiments, the methods of the present invention do not reduce mCD28 levels on immune cells. In some embodiments, the methods of the present invention do not reduce mCD28-mediated immune cell activation. In some embodiments, the methods of the present invention maintain mCD28 levels on immune cells in a subject. In some embodiments, the methods of the present invention increase mCD28 levels on immune cells in a subject.

[0186] In some embodiments, the reduction is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% reduction of sCD28. Each possibility represents a separate embodiment of the present invention. In some embodiments, the reduction is in serum sCD28. In some embodiments, the reduction is in blood levels of sCD28. In some embodiments, the reduction is in sCD28 levels in the tumor microenvironment (TME).

[0187] In some embodiments, the subject's blood includes elevated sCD28 levels. In some embodiments, the subject's blood includes elevated sCD28 levels before reduction. In some embodiments, the level is elevated to above the level of healthy subjects. In some embodiments, the subject's sCD28 level is elevated to at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% above the level of healthy subjects. Each possibility represents a separate embodiment of the present invention. In some embodiments, the level is elevated to above 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45 or 50 ng / ml of blood. Each possibility represents a separate embodiment of the present invention. In some embodiments, the level is increased to above 5 ng / ml. In some embodiments, the level is increased to above 10 ng / ml. In some embodiments, the level is increased to above 20 ng / ml. In some embodiments, the blood of the subject comprises at least 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45 or 50 ng sCD28 / ml blood. Each possibility represents a separate embodiment of the present invention. In some embodiments, the blood of the subject comprises at least 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45 or 50 ng sCD28 / ml blood before reduction. Each possibility represents a separate embodiment of the present invention. In some embodiments, the blood of the subject comprises at least 5 ng / ml sCD28. In some embodiments, the blood of the subject comprises at least 10 ng / ml sCD28. In some embodiments, the subject's blood comprises at least 20 ng / ml sCD28. In some embodiments, the subject's blood comprises at least 5 ng / ml sCD28 prior to reduction. In some embodiments, the subject's blood comprises at least 10 ng / ml sCD28 prior to reduction. In some embodiments, the subject's blood comprises at least 20 ng / ml sCD28 prior to reduction.

[0188] In some embodiments, the subject has cancer. In some embodiments, the cancer is a cancer that can be treated with PD-1 / PD-L1 therapy. In some embodiments, the subject has undergone PD-1 / PD-L1 therapy. In some embodiments, the subject is a non-responder to PD-1 / PD-L1 therapy. In some embodiments, the subject has not undergone PD-1 / PD-L1 therapy. In some embodiments, the method of the present invention is performed together with PD-1 / PD-L1 therapy. In some embodiments, the method of the present invention is performed before PD-1 / PD-L1 therapy.

[0189] In some embodiments, the method further comprises administering another immunotherapy to the subject. In some embodiments, the method further comprises administering an immunotherapy based on PD-1 and / or PD-L1. In some embodiments, the other immunotherapy is a restriction point inhibitor. In some embodiments, the restriction point inhibitor is a PD-1 and / or PD-L1 inhibitor. In some embodiments, the restriction point inhibitor is a CTLA-4 inhibitor. In some embodiments, the other immunotherapy is an immunotherapy based on a chimeric antigen receptor (CAR). In some embodiments, the CAR is a CAR-T. In some embodiments, the CAR is a CAR-NK. In some embodiments, the other immunotherapy is a cancer vaccine.

[0190] As used herein, the terms "CAR-T cells" and "CAR-NK cells" refer to engineered receptors that are specific to at least one target protein (e.g., an immunogenic protein with increased expression after using an epigenetic modifier) ​​and transplanted onto immune effector cells (T cells or NK cells). In some embodiments, CAR-T cells have the specificity of monoclonal antibodies transplanted onto T cells. In some embodiments, CAR-NK cells have the specificity of monoclonal antibodies transplanted onto NK- cells. In some embodiments, T cells are selected from cytotoxic T lymphocytes and regulatory T cells.

[0191] CAR-T and CAR-NK cells and their vectors are well known in the art. Such cells target receptor-bound proteins and are cytotoxic to them. In some embodiments, CAR-T or CAR-NK cells target at least one viral protein. In some embodiments, CAR-T or CAR-NK cells target multiple viral proteins. In some embodiments, CAR-T or CAR-NK cells target viral proteins that have increased expression due to contact with an epigenetic modifier.

[0192] The construction of CAR-T cells is well known in the art. In a non-limiting example, monoclonal antibodies to viral proteins can be made, and then the vector encoding the antibody will be constructed. The vector will also include a costimulatory signal region. In some embodiments, the costimulatory signal region includes an intracellular domain of a known T cell or NK cell stimulating molecule. In some embodiments, the intracellular domain is selected from at least one of the following: CD3Z, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD 7, LIGHT, NKG2C, B7-H3, and a ligand specifically bound to CD83. In some embodiments, the vector also includes a CD3Z signaling domain. The vector is then transfected into T cells, for example, by lentiviral infection.

[0193] In some embodiments, cancer is a cancer with sCD28 levels. In some embodiments, cancer includes high sCD28 levels. In some embodiments, elevated and / or high sCD28 levels are at and / or above 5, 6, 7, 8, 9, 10, 12, 14, 15, 17, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or 100 ng / ml levels. Each possibility represents a separate embodiment of the present invention. In some embodiments, cancer includes high sCD28 levels. In some embodiments, elevated and / or high sCD28 levels are at and / or above 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of the levels in healthy subjects. Each possibility represents a separate embodiment of the present invention. In some embodiments, cancer is not breast cancer. In some embodiments, the cancer is selected from melanoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, kidney cancer, gastric cancer, and colorectal cancer. In some embodiments, the cancer is selected from melanoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, kidney cancer, gastric cancer, or colorectal cancer. Each possibility represents a separate embodiment of the present invention.

[0194] Reagent test kit

[0195] By way of another aspect, there is provided a kit comprising at least one agent of the invention or a pharmaceutical composition of the invention.

[0196] In some embodiments, the kit further comprises an immunotherapeutic agent based on PD-1 and / or PD-L1. In some embodiments, the kit comprises a label indicating that the reagent of the present invention is used together with the immunotherapeutic agent based on PD-1 and / or PD-L1. In some embodiments, the kit comprises a label indicating that the therapeutic agent based on PD-1 and / or PD-L1 is used together with the antibody or pharmaceutical composition of the present invention.

[0197] By way of another aspect, a kit comprising a PD-1 and / or PD-L1 based immunotherapeutic agent is provided, comprising a label indicating the use of the PD-1 and / or PD-L1 based therapeutic agent with an antibody or pharmaceutical composition of the invention.

[0198] In some embodiments, the kits of the present invention are used to treat cancer. In some embodiments, the kits of the present invention are diagnostic kits. In some embodiments, the kits of the present invention are used to determine the serum level of sCD28 in a subject in need thereof. In some embodiments, the subject suffers from cancer. In some embodiments, the kits of the present invention are used to determine the suitability of a subject to be treated with an agent or pharmaceutical composition of the present invention. In some embodiments, the kits are used to determine the suitability of a subject to be treated with an anti-PD-1 / PD-L1 based immunotherapy.

[0199] Methods of Reagent Generation

[0200] By another aspect, there is provided a method of producing an agent that inhibits proteolytic cleavage of mCD28 on the surface of a cell, comprising:

[0201] a. obtaining an agent that binds to the extracellular domain of CD28 or a fragment thereof, wherein the agent is less than 100 kDa;

[0202] b. testing the binding of the obtained reagent to mCD28 on the cell surface; and

[0203] c. Selecting a reagent that binds to cell surface mCD28;

[0204] Thereby a reagent is generated which inhibits the proteolytic cleavage of mCD28 on the surface of cells.

[0205] By another aspect, there is provided a method of producing an agent that inhibits proteolytic cleavage of mCD28 on the surface of a cell, comprising:

[0206] d. Cultivating a host cell comprising one or more vectors comprising a nucleic acid sequence encoding an agent, wherein the nucleic acid sequence is a nucleic acid sequence of an agent selected by:

[0207] i. obtaining an agent that binds to the extracellular domain of CD28 or a fragment thereof, wherein the agent is less than 100 kDa;

[0208] ii. testing the obtained reagent for binding to mCD28 on the cell surface; and

[0209] iii. selecting a reagent that binds to cell surface mCD28;

[0210] Thereby a reagent is generated which inhibits the proteolytic cleavage of mCD28 on the surface of cells.

[0211] In some embodiments, the method further comprises testing the ability of a protease that blocks the shearing of mCD28 on the cell surface. In some embodiments, the agent is an anti-shearing agent. In some embodiments, the agent is an anti-shedding agent. In some embodiments, the agent reduces the shedding of sCD28 in a subject. In some embodiments, the agent reduces the shearing of mCD28. In some embodiments, the agent reduces the shearing of mCD28 in a subject.

[0212] In some embodiments, the protease is MMP-2. In some embodiments, the protease is MMP-13. In some embodiments, the protease is ADAM10. In some embodiments, the protease is ADAM17. In some embodiments, the protease is MMP-2, ADAM10, ADAM17, or a combination thereof. MMP-2, MMP-13, ADAM10, ADAM17, or a combination thereof.

[0213] As used herein, the term "extracellular domain of CD28" refers to the N-terminal portion of CD28 that reaches before the transmembrane domain. In some embodiments, the extracellular domain of CD28 is sCD28. In some embodiments, the extracellular domain of CD28 is CD28a. In some embodiments, the extracellular domain of CD28 is the CD28 stem domain. In some embodiments, the extracellular domain of CD28 includes the stem domain of CD28. In some embodiments, the extracellular domain of CD28 includes or consists of the following sequence: NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQID NO: 28). In some embodiments, the extracellular domain of CD28 or a fragment thereof is dimer. In some embodiments, the extracellular domain of CD28 or a fragment thereof is monomeric. In some embodiments, the extracellular domain of CD28 or a fragment thereof is dimeric or monomeric.

[0214] As used herein, "fragment" refers to a partial polypeptide that constitutes a portion of a larger protein or protein domain. In some embodiments, the fragment comprises at least 10, 20, 30, 40 or 50 amino acids. Each possibility represents a separate embodiment of the present invention. In some embodiments, the fragment comprises at most 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids. Each possibility represents a separate embodiment of the present invention. In some embodiments, the reagent that obtains a fragment that binds to the extracellular domain of CD28 is a reagent that obtains specific binding to the CD28 stem domain.

[0215] In some embodiments, the method further comprises measuring mCD28 downstream signaling in the presence of the obtained agent and selecting at least one agent that neither substantially agonizes nor substantially antagonizes mCD28 signaling. In some embodiments, the selection is to select at least one agent that does not antagonize mCD28 signaling. Those skilled in the art will appreciate that for cancer treatment, agonizing CD28 signaling may not be harmful, but antagonizing signaling can be counterproductive.

[0216] In some embodiments, the ability of the test reagent to block shearing includes measuring sCD28 in the serum of activated immune cells in the presence or absence of the reagent. In some embodiments, the ability of the test reagent to block shearing includes mixing the reagent, protease and the extracellular domain of CD28 or a fragment thereof including a shearing site. In some embodiments, the test further includes sequencing the extracellular domain of CD28 or a fragment thereof to check for truncation and / or shearing. In some embodiments, the test further includes running the extracellular domain of CD28 or a fragment thereof on a sufficiently sensitive gel to measure the size change caused by shearing. In some embodiments, the test further includes measuring the production of sCD28 from cells expressing mCD28 in the presence of the reagent and protease.

[0217] In some embodiments, obtaining the reagent comprises immunizing a shark or camelid with the CD28 extracellular domain or a fragment thereof and collecting antibodies from the immunized organism. In some embodiments, obtaining the reagent comprises screening a library of reagents that bind to the CD28 extracellular domain or a fragment thereof and selecting a binding reagent.

[0218] In some embodiments, collecting antibodies comprises extracting B cells from the spleen of an immunized shark or camelid. In some embodiments, B cells are fused with melanocytes to produce hybridomas. In some embodiments, antibodies are collected from the culture medium of hybridomas. In some embodiments, obtaining reagents comprises immunizing an organism with a CD28 extracellular domain or a fragment thereof, and collecting antibodies from the immunized organism. In some embodiments, the organism is a mouse. In some embodiments, the organism is selected from rabbits, mice, rats, sharks, camelids, chickens, goats, and bacteriophages. In some embodiments, the camelids are selected from camels and llamas. In some embodiments, collecting comprises drawing blood. In some embodiments, collecting comprises:

[0219] e. Extracting B cells from the spleen of an immune organism;

[0220] f. fusing the extracted B cells with myeloma cells to produce hybridomas; and

[0221] g. Collecting antibodies from hybridomas.

[0222] In some embodiments, obtaining a reagent includes screening a library of reagents that bind to the CD28 extracellular domain or a fragment thereof and selecting the reagents so bound. In some embodiments, the library is a phage display library. In some embodiments, the library is an immune library derived from spleen B cells. In some embodiments, the library is an IgG library. In some embodiments, the library is a Fab library. In some embodiments, the library is a VHH antibody library. In some embodiments, the library is a single chain, single domain or nano antibody library. In some embodiments, obtaining a reagent includes sequencing a reagent. In some embodiments, obtaining a reagent includes generating a recombinant form of a reagent. In some embodiments, selecting a reagent includes sequencing a reagent. In some embodiments, selecting a reagent includes generating a recombinant form of a reagent. In some embodiments, a recombinant form is generated from the sequence of a reagent. In some embodiments, the method further includes a humanized reagent.

[0223] The nucleic acid molecules encoding the reagents expressed in cells are well known to those skilled in the art. It can be carried out by many methods such as transfection, viral infection or direct changes in the cell genome. In some embodiments, the gene is present in an expression vector, such as a plasmid or a viral vector. An example of such an expression vector comprising p16-Ink4a is the mammalian expression vector pCMV p16 INK4A available from Addgene.

[0224] The vector nucleic acid sequence generally contains at least an origin of replication for propagation in cells and optionally additional elements, such as a heterologous polynucleotide sequence, expression control elements (e.g., promoters, enhancers), selectable markers (e.g., antibiotic resistance), polyadenine sequences.

[0225] The vector can be a DNA plasmid delivered by a non-viral method or a viral method. The viral vector can be a retroviral vector, a herpes virus vector, an adenoviral vector, an adeno-associated virus vector or a poxvirus vector. The promoter can be active in mammalian cells. The promoter can be a viral promoter.

[0226] In some embodiments, the nucleic acid sequence encoding the agent is operably linked to a promoter. The term "operably linked" is intended to indicate that the target nucleotide sequence is linked to one or more regulatory elements in a manner that allows the nucleotide sequence to be expressed (e.g., in an in vitro transcription / translation system or in a host cell, when the vector is introduced into a host cell).

[0227] In some embodiments, the vector is introduced into the cell by standard methods, including electroporation (e.g., as described in From et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985)), heat shock, infection with a viral vector, high-speed ballistic penetration of small particles with nucleic acids within the matrix or on the surface of beads or particles (Klein et al., Nature 327. 70-73 (1987)), and / or the like.

[0228] As used herein, the term "promoter" refers to a group of transcription control modules that are clustered around the start site of RNA polymerase, RNA polymerase II. A promoter is composed of discrete functional modules, each of which consists of approximately 7-20 bp DNA and contains one or more recognition sites for transcriptional activators or repressors.

[0229] In some embodiments, the transcribed sequence is transcribed by RNA polymerase II (RNAP II and Pol II). RNAPII is an enzyme present in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA.

[0230] In some embodiments, mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(±), pGL3, pZeoSV2(±), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81, which are available from Invitrogen; pCI, which is available from Promega; pMbac, pPbac, pBK-RSV and pBK-CMV, which are available from Strategene; pTRES, which is available from Clontech, and derivatives thereof.

[0231] In some embodiments, the present invention uses expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papilloma virus include pBV-1MTHA, and vectors derived from Epstein-Barr virus include pHEBO and p2O5. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and any other vectors that allow expression of proteins under the guidance of SV-40 early promoter, SV-40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter or other promoters effective in eukaryotic cells.

[0232] In some embodiments, recombinant viral vectors, which have advantages such as lateral infection and targeting specificity, are used for in vivo expression. In one embodiment, lateral infection is, for example, inherent in the life cycle of retroviruses, and is the process by which a single infected cell produces many progeny virions that sprout and infect neighboring cells. In one embodiment, the result is that large areas are rapidly infected, most of which were not initially infected by the original viral particles. In one embodiment, viral vectors that cannot be spread laterally are produced. In one embodiment, this feature may be useful if the desired purpose is to introduce a specific gene into only a local number of target cells.

[0233] The expression vector of the present invention can be introduced into cells using a variety of methods. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et al., [Biotechniques 4 (6): 504-512, 1986], and include, for example, stable or transient transfection, liposome transfection, electroporation and infection of recombinant viral vectors. Additionally, see US Patent Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

[0234] It will be appreciated that, in addition to the elements necessary for transcription and translation of the inserted coding sequence (encoding a polypeptide), the expression constructs of the invention may also include sequences engineered to optimize stability, production, purification, yield or activity of the expressed polypeptide.

[0235] By another aspect, there is provided an agent produced by the method of the present invention.

[0236] By another aspect, a pharmaceutical composition is provided, which includes an agent produced by the method of the present invention and a pharmaceutically acceptable carrier, excipient or adjuvant.

[0237] As used herein, the term "about" when combined with a value refers to plus or minus 10% of the reference value. For example, a length of about 1,000 nanometers (nm) refers to a length of 1,000 +- 100 nm.

[0238] Note that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "polynucleotide" includes a plurality of such polynucleotides, and reference to a "polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. Note further that the claims may be drafted to exclude any optional element. Therefore, this statement is intended to serve as antecedent basis for the use of terminology such as "solely," "only," and the like in connection with the recitation of claim elements, or the use of a "negative" limitation.

[0239] In those cases where a convention similar to "at least one of A, B, and C, etc." is used, generally speaking, such construction is intended to be in the sense that one skilled in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include, but is not limited to, systems having: A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). One skilled in the art would further understand that substantially any transitional word and / or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both of the terms. For example, the phrase "A or B" would be understood to include the possibility of "A" or "B" or "A and B."

[0240] It should be understood that certain features of the invention described in the context of separate embodiments for the sake of clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention described in the context of a single embodiment for the sake of brevity may also be provided individually or in any suitable sub-combination. All combinations of embodiments related to the present invention are specifically included in the present invention and are disclosed herein as if each combination was individually and explicitly disclosed. In addition, all sub-combinations of various embodiments and their elements are also specifically included in the present invention and are disclosed herein as if each such sub-combination was individually and explicitly disclosed herein.

[0241] Other objects, advantages and novel features of the present invention will become apparent to those skilled in the art by examining the following examples, which are not intended to be limiting. In addition, each of the various embodiments and aspects of the present invention as described above and claimed in the claims section finds experimental support in the following examples.

[0242] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section herein find experimental support in the following examples.

[0243] Example

[0244] Generally, the nomenclature used herein and the laboratory procedures used in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. These techniques are explained in detail in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM, ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); the methods described in U.S. Patent Nos. 4,666,828, 4,683,202, 4,801,531, 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, JE, ed. (1994); "Culture of Animal Cells-A Manual of Basic Technique" by Freshney, Wiley-Liss, NY (1994), third edition; "Current Protocols in Immunology" Volumes I-III Coligan JE, ed. (1994); Stites et al.(eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization-A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated herein by reference. Other general references are also provided in this document. .

[0245] Materials and methods

[0246] Antibody – Commercial mouse monoclonal anti-CD28 clone #CD28.2 (Biolegend, Cat.No.302902) and FITC conjugated (Biolegend, Cat.No.302906). Goat polyclonal anti-CD28 (R&D system, Cat.No.AF-342-PB). FITC conjugated anti-human PD-L1 (BD bioscience, Cat.No.558065). APC conjugated anti-human PD-L2 (Biolegend, Cat.No.345508). PE conjugated anti-human IDO (R&D system, Cat.No.IC6030P). Goat anti-mouse IgG Alexa Fluor 647 (Biolegend, Cat.No.405322). Donkey anti-human IgG (H+L) Alexa Fluor647 (Jackson immune research, Cat.No.709-605-149). Goat anti-mouse IgG HRP (Jacksonimmune research, Cat. No. 115-035-071). Anti-human CD3 clone OKT3 (Biolegend, Cat. No. 317304). Anti-human PD-1 pembrolizumab (MK-3475). Human IgG (Sigma, Cat. No. I4506).

[0247] Isolation of VHHs targeting the stalk region of the human CD28 receptorThe genetic code of peripheral blood B cells, derived from a non-immune Llama used for the first experiment, was used to construct a phage library consisting of particles expressing individual VHHs as fusion proteins with a C-terminal His6-Myc tag. The library used for the first experiment was used to select nanobodies with the ability to bind to the stem region of human CD28. Biotinylated recombinant CD28-Fc chimeras or oxidized dimeric peptides with the sequence of "HVKGKHLCPSPLFPGPSKP (SEQ ID NO: 10)" were screened, with biotin added at the C-terminus. Each antigen was bound to streptavidin magnetic beads, which were blocked by skim milk. The same antigen was used in three consecutive selection rounds, changing the phage input amount and antigen concentration, and the phage was selected in solution. Blocked beads without antigen were used as controls. Elution of bound phages was performed with trypsin for 20 minutes. The enrichment rate during in-solution selection was calculated as the ratio between the number of phages eluted from the CD28 antigen selection conditions and the number of phages eluted from the antigen-free selection conditions. 279 selected output individual phage monoclones, in either phage or periplasmic form, were validated for antigen binding by ELISA and characterized for binding to membrane CD28 by flow cytometry. 72 clones showed specific binding to the stem region peptide in the periplasmic form, 22 were demonstrated to have unique CDR sequences, while only 6 were found to belong to unique CDR3 families. The 6 VHHs were produced as recombinant proteins in CHO cells with a C-terminal His tag and evaluated for anti-shedding activity and cell binding. Transfection – CD28wt (encoding full-length CD28 transcript) plasmid was generated by cloning the DNA sequence into the pcDNA3.1 vector. Transfection was performed using Jet Pei transfection reagent (PolyPlus transfection). Stable transfectants were selected in medium containing G418.

[0248] ELISA- Commercial ELISA kits were used to quantify the amount of: interferon-γ (Biolegend, Cat. No. 430103), human interleukin 2 (Biolegend, Cat. No. 431802), human interleukin 6 (Biolegend, Cat. No. 430502), human interleukin 10 (Biolegend, Cat. No. 430603), human tumor growth factor β1 (Biolegend, Cat. No. 436708), human interleukin β1 (Biolegend, Cat. No. 437004) and human CD28 (R&D system, Cat. No. DY342). Cell proliferation and viability (MTT assay) were performed according to the manufacturer's instructions (Roche, Cat. No. 11465007001). Kynurenine (IDO activity) ELISA kit (ImmuSmol, Cat. No. BA E-2200) was performed according to the manufacturer's instructions.

[0249] CD28 stalk region binding assay - Biotin-conjugated wild-type or L145K CD28 stalk region dimer peptides were immobilized on neutravidin-coated ELISA maxi-sorb plates. VHH clones were serially diluted (0.2-5 μg / mL) and bound VHHs were detected using an anti-His tag-HRP conjugated antibody and visualized using TMB.

[0250] Cytokine multiplex - Multiple cytokines were assessed simultaneously using ProcartaPlex (Invitrogen, Cat. No. PPX-07-MXXGPY2) on the Magpix system (Millipore).

[0251] Flow cytometry – Generally, cells were kept on ice during all steps. Before staining, 5×10 5 Cells were incubated for 15 min per cell. Antibodies were used at the concentration recommended by the manufacturer and incubated for 30 min in the dark. Incubation was performed in a volume of 100 μL in a 96-well U-bottom plate. Cells were washed twice with 200 μL of FACS buffer and transferred to FACS tubes in 150 μL of FACS buffer for analysis. Cells were analyzed on a Gallios flow cytometer (Beckman Coulter) using Kaluza for Gallios flow cytometer acquisition software.

[0252] Cell lines and isolation of human immune cells– Jurkat leukemic T-cell lymphoblastoid cell line clone E6.1 and SCC-25 tongue squamous cell carcinoma were obtained from ATCC. PBMCs were isolated from fresh blood samples of healthy donors using standard lymphocyte separation medium (MBP, Cat. No. 850494). Negative selection was performed using Rossette SEP TM CD3 T cells were isolated from fresh blood samples of healthy donors using the human T cell enrichment kit (STEMCELL, Cat. No. 15061). TM CD4 cells were isolated from fresh blood samples of healthy donors using the human T cell enrichment kit (STEMCELL, Cat. No. 19059). TM Mononuclear cells were isolated from fresh blood samples of healthy donors using the Human T Cell Enrichment Kit (STEMCELL, Cat. No. 17952). All cells were grown in complete RPMI-1640 medium supplemented with 10% HI-FCS and pen / strep mixture.

[0253] CD86 blocking FACS - At room temperature, 0.5 × 10 6 HEK293 cells were incubated with 2 μg / mL CD86-Fc (R&D systems, Cat. No. 141-B2) without or with anti-CD28 antibody (CD28.2, 10 μg / mL) or VHH clone (30 μg / mL) for 30 min. Cells were washed and secondary binding was performed on ice for 20 min using anti-human heavy and light chain antibodies conjugated to fluorophores at a dilution of 1:5000.

[0254] Dendritic cell differentiation – Monocytes were cultured in RPMI medium at 1×10 6 / mL, with growth factors supplemented on days 3 and 6. Immature dendritic cells (iDCs) were induced with 50ng / mL GM-CSF and 20ng / mL IL-4 for 6 days. When required, iDCs were further differentiated into mature dendritic cells by adding 100ng / mL LPS for 48 hours. The resulting cell populations were tested for designated phenotypes by FACS analysis of relevant markers and secretion analysis of characteristic cytokines.

[0255] Metalloproteinases- Commercial recombinant human metalloprotease MMP-2 from Anaspec (Cat. No. AS-72005) or R&D system (Cat. No. 902-MP) was used. Commercial recombinant human metalloprotease MMP-13 was purchased from R&D system (Cat. No. 511-MM). Pro-MMP2 and Pro-MMP-13 were activated with 1 mM aminophenylmercuric acetate (APMA) at 37°C for 1-2 hours according to the manufacturer's protocol.

[0256] Protease inhibitors – Protease inhibitors were added at the beginning of each experiment at the indicated concentrations. In the one-week experiments, another portion of inhibitors was added after 3 days at the final concentration. The protease inhibitors used were TAPI-1 (Cayman, Cat. No. 18505), GM6001 (Santa Cruz, Cat. No. SC-203979), TMI-1 (Sigma, Cat. No. PZ0336) and GI254023X (Sigma, Cat. No. SML0789). The protease mixture mentioned therein consisted of a mixture of TAPI-1 and GM6001 in an equimolar ratio.

[0257] Synthetic peptides – The substrate peptide of the final form “DYKDDDDKGGGGGHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 41)-biotin” was designed to include the amino acid sequence of the human CD28 stem region (His134-Pro152) between the N-terminal cMyc tag and the C-terminal biotin conjugation followed by a 5-glycine sequence. The peptide was custom synthesized by Gencust Europe. The cysteine ​​residue at position 141 was used to generate a dimeric peptide via a disulfide bond. A CD28 stem region peptide with a mutation at the cleavage site, a leucine to lysine substitution at position 145, was similarly synthesized, which had the final form “DYKDDDDKGGGGGHVKGKHLCPSPKFPGPSKP (SEQ ID NO: 42)-biotin”.

[0258] In vitro shear assay- 50 ng of purified recombinant MMP-2 or MMP-13 were incubated with 0.125 μM dimeric c-Myc-tagged and biotinylated substrate peptides in the presence or absence of MMP inhibitors (TMI-1, 50 nM), M9 Fab or the indicated VHH clones at various concentrations (0.4-10 μg / mL) for 5 hours. The assay was performed in 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35, pH 7.5. After 5 hours, the shearing reaction mixture was diluted to a final peptide concentration of 1 nM and loaded on a neutravidin plate to bind the peptide. After incubation at room temperature for 1 hour, the plates were washed and detection of uncleaved peptides was performed using an anti-cMyc antibody conjugated to HRP.

[0259] CD4 T cells or Jurkat PHA activation of T cell lines for production of soluble CD28 – 1×10 5 Jurkat cells or CD4 T cells were incubated with the indicated concentrations of phytohemagglutinin (Sigma, Cat. No. L8902) and various protease inhibitors for an additional 5 days (Jurkat) or 7 days (CD4 T cells).

[0260] SEB or CMV activation of PBMCs for production of soluble CD28 - 0.3 × 10 cells / well were stimulated with 0.5 ng / mL SEB (Sigma, Cat. No. S4881) at 37°C in a 48-well plate with / without various protease inhibitors at the indicated concentrations. 6 PBMCs were maintained for 5-7 days. Alternatively, 0.1 × 10 6 For CMV stimulation, 0.5×10 PBMCs were stimulated with 0.5 μg / mL CMV peptide peptivator (Milteny Biotec, Cat. No. 130-093-435) in 96-well plates at 37°C with / without various protease inhibitors at the indicated concentrations. 6 PBMCs were maintained for 2-5 days. For serial shedding experiments, PBMCs were stimulated with SEB or CMV for 24 hours in 24-well plates, cells were removed and washed three times with RPMI without stimulants, and plated again in 96-well plates. Samples were removed at the indicated times and kept in a frozen condition until soluble CD28 was examined.

[0261] Cellular assay to evaluate the anti-shedding activity of VHHs - For SEB activation of PBMCs, 0.1×10 PBMCs were stimulated with 2 ng / mL SEB (Sigma, Cat. No. S4881) at 37°C in 96-well plates with or without various treatments at the indicated concentrations. 6PBMCs were maintained for 5-7 days. For PHA-activated T cells, 0.1×10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 6 For the spontaneous CD28 shedding assay on HEK, 0.1 × 10 CD4 T cells were incubated in 96-well plates at 37 °C with or without various treatments at the indicated concentrations. 5 HEK cells for 48 hours.

[0262] Mixed lymphocyte reaction – 1×10 5 Immature DCs and 5×10 5 The isolated autologous CD3 T cells were mixed for 6 days.

[0263] SEB or CMV stimulation assay with ectopic recombinant human CD28, human CTLA-4, and human CD80 – For CMV stimulation, 0.5×10 cells were stimulated with 0.5 μg / mL CMV peptide activator (Milteny Biotec, Cat. No. 130-093-435) in 96-well plates at 37°C with / without the indicated concentrations of recombinant human CD28 (R&D system, Cat. No. 342-CD), human CTLA-4 (R&D system, Cat. No. 434-CT), human CD80 (R&D system, Cat. No. 140-B1). 6 PBMCs (from healthy or cancer patient donors) were cultured for 2-5 days. For SEB setup, 1×10 cells were cultured with 0.5 ng / mL Staphylococcal enterotoxin B (SEB) (Sigma, Cat. No. S4881) in the presence of the indicated concentrations of rec. human CD28. 5 PBMCs were cultured for 72 hours. Where indicated, anti-PD1 or human IgG was added at a final concentration of 5 μg / mL.

[0264] Autologous monocyte CD3 MLR -0.5×10 6 T cells were compared with 0.5 × 10 from the same CMV-reactive donor. 5 Monocytes were pooled and stimulated with 0.5 μg / mL CMV peptide activators for 6 days at 37°C with or without treatment at the indicated concentrations.

[0265] Stimulation of monocytes with recombinant human CD28– 1.5×10 cells were plated in 24-well plates in RPMI medium (R&D system, Cat. No. 285-IF) with 100-100 U / ml IFNγ in the presence of recombinant human CD28 at the indicated concentrations. 6 Monocytes 48 hours. The resulting cell populations were tested for the indicated phenotypes by FACS analysis of relevant markers (IDO, PD-L1 and PD-L2) and by analyzing the secretion of a characteristic cytokine (IL-6).

[0266] T cell stimulation with OKT3 – 0.1 × 10 cells were stimulated with the indicated amount of anti-CD3 clone OKT3 at 37°C 6 Isolated CD3 T cells (from healthy donors) were maintained for 48-72 hours. Where indicated, recombinant human CD80-Fc (2 μg / mL, R&D system) was added in soluble form. Antibodies or VHHs were added to CD28 or controls in soluble form at the indicated concentrations.

[0267] Co-culture of SCC-25 cancer cell line with monocytes in a transwell-based assay – 4×10 4 SCC-25 cells were plated on 1×10 5 monocytes were cultured in the bottom of 24-well plates with or without the indicated treatments in serum-free starvation medium for 4 days.

[0268] Detection of soluble human CD28 in plasma of cancer patients - 20 frozen plasma samples of each of 10 different cancer indications and healthy donors were purchased from DxBiosamples (San Diego, CA, USA). Plasma samples were diluted 1:20 and analyzed for soluble human CD28 by ELISA. Samples with high CD28 were analyzed again at appropriate dilutions.

[0269] Direct CD28 EIA - Unless otherwise discussed, screening was performed using Corning High Binding Plates or equivalent. 200-300 ng of human CD28-Ig chimera (R&D, Cat. No. 342-CD), mouse CD28-Ig chimera (R&D, Cat. No. 483-CD) or BSA-conjugated dimeric peptide consisting of the amino acid sequence of the CD28 stem region (Gly137-Pro152) was coated per well. Plates were blocked with 5% milk or 1% casein in PBS for 1 hour at room temperature (RT). Plates were washed 3 times with PBST and incubated with the study antibodies after detection with goat anti-mouse HRP Fc at a specific dilution of 1:5000. Positive controls were mouse anti-human CD28 clone 28.2 or mouse sera from immunized mice. Hybridoma supernatant cultures screened were not diluted.

[0270] Antibody sequencing. The antibodies were provided to Rapid Novor for amino acid sequencing. Sequencing was performed using standard methods, which briefly included LC-MS analysis after enzymatic digestion with six enzymes (pepsin, trypsin, chymotrypsin, elastase, Lys C and AspN). Digestion was performed with disulfide reduction and alkylation. LC-MS / MS analysis was performed using a Thermo-Fisher Q-exactive mass spectrometer. In the heavy and light chains of each antibody, at least 5 peptide scans covered 100% of the amino acid residues with significant supporting fragment ions. CDRs were determined using the Chothia protocol.

[0271] Example 1: Human CD28 undergoes proteolytic shedding during chronic stimulation

[0272] Soluble CD28 (sCD28) was detected by ELISA in cultures of chronically stimulated human PBMCs ( Figure 1 , upper figure). This phenomenon was evident regardless of the nature of the stimulus, artificial (SEB) or physiological (CMV), indicating the robustness of the phenomenon. Treatment with TAPI-1 and GM6001 (a broad MMP and ADAM17 inhibitor) reduced the amount of sCD28 in a dose-dependent manner, so the source of soluble CD28 is shedding of membrane forms ( Figure 1 , above). Figure 2 As can be seen in the figure, the cellular source of shed CD28 is T cells. Chronic stimulation of Jurkat T cell lines or human CD4 T cells from healthy donor peripheral blood with PHA resulted in the production of sCD28 in a dose-dependent manner ( Figure 2 , upper panel). TAPI-1 and GM6001 were used to treat the cells at each PHA concentration ( Figure 2 , upper figure) and at a fixed PHA concentration ( Figure 2 , lower figure) reduced the amount of sCD28 in a dose-dependent manner.

[0273] Treatment with GI254023X (a highly specific ADAM-10 inhibitor) resulted in almost complete inhibition of sCD28 release from activated immune cells in a dose-dependent manner ( Figure 3A , lower figure). Similar results were observed using the ADAM-17-specific inhibitor TMI-1 ( Figure 3B , lower figure). The viability of immune cells was monitored by examining the metabolic activity of cells in culture by MTT assay. The results showed no significant difference between treatment with and without either ADAM inhibitor, suggesting that low sCD28 levels are an artifact of cell death caused by blocking protease activity rather than by protease inhibitors ( Figure 3A-B, above).

[0274] The production of sCD28 was also validated in more physiological systems. First, using isolated autologous dendritic cells and CD4 T cells that mimic physiological stimulation of T cells by antigen-presenting cells. When the two cell populations were mixed, the rise in sCD28 was evident and became even more prominent when CMV was added to the culture ( Figure 4A ). This suggests that human CD28 protein undergoes proteolytic shedding when chronic stimulation occurs.

[0275] Next, CMV peptide ( Figure 4B ) or SEB( Figure 4C ) to stimulate human PBMCs for 24 hours. Then, the cells were washed to remove the stimulant and plated again without any stimulation for various time periods. The culture medium was then examined for the presence of sCD28. Over time, the accumulation of sCD28 was clearly visible. In addition, the accumulation depended on the activity of ADAM-10 and ADAM-17, such as Figure 4D Addition of different concentrations of a specific inhibitor, after SEB stimulation, led to a decrease in the amount of sCD28 quantified after 120 hours. This study could explain the presence of high amounts of soluble CCD28 in the blood of patients, since CD28 shedding occurs upon primary activation of T cells and does not necessarily require continuous or repeated stimulation.

[0276] Example 2: Soluble human CD28 has immunosuppressive activity

[0277] like Figure 1 As can be seen in the figure below, reducing the levels of sCD28 using a protease inhibitor cocktail directly correlated with an increase in T cell activation, as shown by secreted IFNγ levels, indicating that sCD28 has an immunosuppressive function. Increasing the concentration of the protease inhibitor cocktail resulted in lower levels of sCD28 in the cell culture medium, and these lower levels of sCD28 were negatively correlated with higher levels of secreted IFMγ. To further explore the immunosuppressive effects of sCD28, recombinant human CD28 lacking the transmembrane and cytoplasmic domains was added to CMV-stimulated human PBMC cultures. This resulted in a dose-dependent inhibition of IFNγ secretion ( Figure 5 This immunosuppressive effect was observed in different human PBMC donors, confirming the robustness of this signaling axis blocked by sCD28.

[0278] At the same time, interleukin-6 secretion ( Figure 6 and 7A ) and interleukin-10 ( Fig. 7A) were evident. These cytokines reportedly exhibit suppression of immune effector activity (IL-10) and tilt the immune system toward a type 2 immune response that can support cancer proliferation and angiogenesis through STAT-3 signaling (IL-6). In addition, a comparison with soluble CTLA-4 (mimicking abatacept - a registered therapy for autoimmune disorders) was performed and revealed an overall similar effect on the immune system in terms of cytokine secretion profiles ( Fig. 7A ).

[0279] Next, human PBMCs were stimulated with SEB (1 ng / mL) in the absence or presence of recombinant human CD28. Human IgG was used as a control. The S3 Live-Cell monitors markers of immune activation and takes images every 12 hours. Figure 7C As can be seen in the results, SEB had essentially no effect on lymphocytes in the presence of recombinant human sCD28. It is well known that antigen-presenting cells (APCs) aggregate with each other and with other cell types during in vitro immune responses, and that aggregation is essential for antigen-specific activation of resting lymphocytes. During SEB immunization, soluble CD28 appeared to reduce the amount and size of aggregates formed, meaning that it inhibited the first step in T-cell-specific activation of APCs.

[0280] Similar results were observed when isolated autologous monocytes and CD3 T cells were co-cultured in a mixed lymphocyte reaction (MLR). The mixed cells were stimulated with CMV peptide (0.5 μg / mL) for 5 days with or without increasing concentrations of recombinant human sCD28. Again, sCD28 was found to inhibit IFNγ secretion while increasing the secretion of IL-1B, TGFβ, and IL-10 ( Figure 7B ).

[0281] sCD28 has similar immunosuppressive effects on monocytes. Indoleamine 2,3-dioxygenase (IDO) has been implicated in immunoregulation through its ability to catabolize the essential amino acid tryptophan. It is expressed by different immune cells and also by many cancer cells. Tryptophan deficiency inhibits the maturation and proliferation of T lymphocytes, while kynurenine, the end product of tryptophan catabolism, is also known as an immunosuppressive metabolite that promotes immune tolerance in various physiological and pathological conditions. To test the effect of sCD28 on IDO, isolated human monocytes were stimulated with IFNγ (1000 U / mL) in the presence of control human IgG or recombinant human CD28 (10 μg / mL) for 48 h. After incubation, monocytes were intracellularly stained for human IDO ( Fig. 7ETo facilitate intracellular staining, cells were fixed and permeabilized using the BD Cytofix / Cytoperm Buffer Kit. IDO activity of the culture media from different treatments was assessed using the ImmuSmol-specific kynurenine ELISA kit ( Fig.7D ). sCD28 strongly enhances IDO expression in monocytes.

[0282] Furthermore, sCD28 was surprisingly found to be a potent inhibitor of anti-PD1 immunotherapy. MK-3475 (pembrolizumab or Keytruda, Merck) is an approved drug with unprecedented efficacy in multiple cancer indications. Its addition to PMBC cultures increased pro-inflammatory cytokine secretion (IFNγ and IL-2), but the presence of sCD28 completely abolished this immune activation effect ( Fig. 8A ).

[0283] Similar results were observed again in the MLR setting. MLR was run as before only with and without sCD28 and with and without anti-PD1 antibody (MK3475, 5 μg / mL) ( Figure 8B As expected, MK-3475 increased IFNγ secretion and decreased TGFβ secretion. Notably, the effect of MK-3475 was significantly reduced in the presence of sCD28.

[0284] To elucidate the mechanism by which sCD28 inhibits the pro-activating effects of anti-PD-1 therapy, the expression of PD-1 ligands on immune cells in the presence of sCD28 was examined. Isolated human monocytes were stimulated with IFNγ (1000 U / mL) in the presence of control human IgG (10 μg / mL) or recombinant human CD28 (10 μg / mL) for 48 hours. After incubation, expression of PD-L1 ( Figure 8C , left) and PD-L2( Figure 8C , right) for monocyte staining. Both ligands were upregulated on monocytes cultured with sCD28, suggesting a possible way in which sCD28 may circumvent the effects of anti-PD-1 immunotherapy.

[0285] Example 3: Soluble human CD28 was found in the plasma of cancer patients

[0286] Levels of sCD28 in cancer have only been shown in a few breast cancer patients and were found to be only slightly higher than those observed in healthy individuals ((Isitmangil, G., In vivo, 2016). Although the authors suggested that sCD28 could be used as a marker for breast cancer, no functional relationship was suggested. Now that it is known that soluble CD28 may actually enhance cancer evasion of the immune system, an investigation was conducted on 220 samples covering 10 different cancer indications and 20 samples from healthy donors. The investigation found high sCD28 levels in several cancers, sometimes several orders of magnitude higher than those seen in healthy controls and even breast cancer patients ( Fig. 9A In fact, when compared to sCD28 levels found in some patients with melanoma, colorectal cancer, non-small cell lung cancer, and head and neck cancer, levels in breast cancer patients appeared to be comparable to those in healthy individuals.

[0287] To further elucidate the role of sCD28 in cancer, PBMCs were isolated from cancer patients with different indications. The cells were stimulated for 3 days with SEB (5 ng / mL) alone, or with MK-3475, with recombinant human sCD28, or with a combination of both molecules. In the presence of sCD28, the concentration of human IFNγ in the supernatant from all donor cells was greatly reduced, even in the presence of MK-3475 ( Fig. 9B ). In fact, sCD28 abolished the effect of MK-3475.

[0288] Next, cells of the head and neck cancer cell line SCC-25 were incubated alone or with monocytes in a transwell assay. SCC-25 cells grown alone were administered IL-6 as a positive control and indeed cell proliferation was increased, as measured by MTT ( Fig. 9C , top) and measured by % confluence ( Fig. 9C , below). Growing cancer cells in the presence of monocytes also increased proliferation, but by far the greatest increase observed was with co-cultures that included sCD28. This data further supports that sCD28 has a pro-oncogenic role.

[0289] Example 4: sCD28 inhibits CD80-Fc efficacy

[0290] CD80 is one of the two major ligands of mCD28 along with CD86. The extracellular domain of CD80 fused to the Fc portion has been used as an immunostimulatory molecule and is being studied as a cancer therapy. To examine the effect of CD28 on the efficacy of CD80-Fc, isolated CD3 human T cells were stimulated with plate-bound anti-CD3 antibody (OKT3, 2 μg / mL) in the presence of 2 μg / mL soluble recombinant human CD80-Fc. As expected, CD80-Fc increased IFNγ secretion. However, the addition of sCD28 counteracted the secondary activation effect of CD80-Fc ( Fig. 10A Similarly, when isolated PBMCs were stimulated with CMV peptide for 3 days and then incubated with sCD28, increasing amounts of CD80-Fc were required to generate the expected immune response ( Fig. 10B ).

[0291] Example 5: Effect of sCD28 on cancer in vivo

[0292] Because mice do not splice mCD28, the role of sCD28 cannot be easily examined in mouse models. The closest option is to administer recombinant sCD28 to mice to mimic a situation where sCD28 levels are elevated. This was studied in the H22 syngeneic mouse model. Balb / c fully immunocompetent mice received allogeneic transplants of H22 hepatocellular carcinoma cells. The cells grew even in fully immunocompetent mice, and the addition of anti-PD-1 therapy almost completely abolished tumor growth ( Fig.11A When recombinant human sCD28 was added, the effects of anti-PD-1 therapy were almost completely abolished in both mice ( Fig. 11B ). This suggests that in some subjects, increased levels of sCD28 can have a highly deleterious effect on cancer progression.

[0293] Example 6: Characterization of Anti-Shedding Antibody-Based Reagents

[0294] The discovery that human CD28 undergoes proteolytic processing by ADAM10 and ADAM17 prompted the examination of candidate regions of its polypeptide sequence, showing potential susceptibility to proteolytic release. Studies have shown that ADAM10 and ADAM17 prefer leucine, valine and aromatic residues at the P1' site. The most attractive sequence region in human CD28 is the stem segment, ranging from histidine 134 to proline 152 (SEQ ID NO: 10 (HVKGKHLCPSPLFPGPSKP)), connecting the globular IgV domain to the transmembrane region. This region contains a total of 3 leucine and valine residues, as well as a phenylalanine residue, and is not expected to have any secondary structural elements that may hinder the entry of proteases. It is worth noting that the stem region also contains cysteine ​​141, which forms an inter-disulfide bond that promotes the homodimerization of CD28. In order to generate antibodies or antibody fragments that specifically bind to the CD28 stem region and may block different proteases from entering the shed CD28, while avoiding any damage to the CD28 oligomeric structure and function, CD1 mice were immunized with a dimeric peptide that mimics the CD28 stem region. The peptide sequence used for immunization is SEQ ID NO: 29, GKHLCPSPLFPGPSKPK, with a C-terminal lysine added to have a free amino group to allow conjugation with KLH or BSA using hydrazide chemistry. Conjugation was performed between the hydrazide-terminated CD28 peptide and S-4FB-modified BSA, which produces free aldehydes for site-specific conjugation. Dimerization was confirmed by running the peptide on a non-denaturing gel.

[0295] An antibody with high binding affinity to recombinant human CD28 as measured by direct CD28 EIA was discovered. The antibody was designated M9 and the sequence of the antibody is provided above. Serial dilutions of antibody M9 were used to confirm its specific binding to recombinant human sCD28 and the stem region peptide ( Fig. 12A Interestingly, while the antibody was able to detect recombinant human sCD28, it was unable to detect sCD28 that was actually shed from immune cells ( Fig. 12B ). This strongly suggests that the antibody binds at the cleavage site and that the deisotope to which it binds is incomplete in the cleaved form.

[0296] Next the ability of the antibodies to bind to mCD28 on the cell surface was investigated. In order to reduce shedding of sCD28 from cells, the antibodies needed to actually bind to the membrane form of the protein and not just the recombinant protein in solution. HEK293 cells overexpressing full-length human CD28 were analyzed. Mouse CD28 does not appear to be sheared into a soluble form (activated mouse splenocytes do not appear to produce sCD28), and therefore the human protein had to be studied. The cells were analyzed by flow cytometry using the M9 antibody and the CD28.2 antibody as a positive control. Surprisingly, M9 did not appear to bind to surface mCD28 ( Fig. 12C). This is probably due to steric hindrance and limited access to the stem region when it is adjacent to the membrane.

[0297] Example 7: Single domain antibody inhibits shedding of sCD28 from the cell surface

[0298] Small reagents were designed that bind to mCD28 on the cell surface and block the shedding of sCD28. While the size of a full-size antibody is approximately 150 kDa, the Fab fragment derived from the antibody has a size of approximately 50 kDa, while single-chain antibodies (also called single-chain variable fragments, scFvs) have a size of approximately 25 kDa, and single-domain antibodies (also called VHH antibodies, scFvs, and DARPins) have a size of only 12-15 kDa.

[0299] Single domain antibodies were isolated using a phage library of llama-derived VHHs for the first time. The library consisted of VHH sequences taken from non-immune llamas for the first time, i.e., B cells were extracted and the entire available repertoire of VHH CDRs was sequenced. These CDRs were introduced into phage to generate the library. The library was screened for the recombinant CD28 extracellular domain and dimer stem region using ELISA and flow cytometry to find antibodies that specifically bind to the stem region of human C28. The VHH sequences found to specifically bind to the stem region of human CD28 are: EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVTVSSAAAHHHHHH (SEQ ID NO:45, clone 2A1); EVQLVESGGGLVQAGGSLRLSCAASGSLFSINAMAWYRQAPGKQRELVAAITSSGSTNYANSVKGRFTVSRDNAKNTMYLQMNSLKPEDTAVYYCVVDEYGSDYWIWGQGTQVTVSSAAAHHHHHH (SEQ ID NO:45, clone 2A1); NO:46, clone 4A4); and QVQLVESGGGLVQAGGSLRLSCAASGSIFSINAMGWYRQAPGKQRERVAAITSGGSTNYADSVKGRFTISRDNAKNTVYLQMNNLEPRDAGVYYCVVDLYGEDYWIWGQGTQVTVSSAAAHHHHHH (SEQ ID NO:47, clone 4A1). VHH was produced as a recombinant protein in CHO cells and then evaluated for cell binding and anti-shedding activity as described below. The His-tag at the C-terminus was used for purification and linked by triple alanine repeats. The CDRs of the three clones studied are provided in Table 1.

[0300] Table 1

[0301] VHH cloning CDR1 (SEQ ID) CDR2 (SEQ ID) CDR3 (SEQ ID) 2A1 INAMG(33) AISGGGDTYYADSVKG(34) DLYGSDYWD(35) 4A4 INAMA (36) AITSSGSTNYANSVKG(37) DEYGSDYWI(38) 4A1 INAMG(33) AITSGGSTNYADSVKG(39) DLYGEDYWI(40)

[0302] Binding of the VHH clones to the human CD28 stem region sequence was first confirmed by ELISA using serial dilutions of the VHH clones ( Fig.13 Binding of membrane human CD28 at the cellular level was confirmed by FACS analysis using labeled VHH clones and HEK cells overexpressing CD28 ( Fig.14). Membrane CD28 binding demonstrated access to the CD28 membrane proximal region. Previous experiments have shown that the size of the agent is critical for access to this region, as full-sized antibodies that can bind to the CD28 stem region peptide are unable to bind to the CD28 stem region on cells. Notably, the VHH clones were unable to bind to the human CD28 stem region sequence with an LK substitution at amino acid residue 145 located within the MMP cleavage site ( Fig.23 ).

[0303] Anti-shedding activity was confirmed at both the peptide and cell levels. ELISA was used to detect the complete human CD28 stalk region dimer peptide to confirm that the VHH clone blocked MMP-2 ( Fig.17 ) and MMP-13( Fig. 22 ) cleavage of the human CD28 stem region. Although M9 Fab showed the ability to block MMP-2 cleavage of the CD28 stem region peptide, it was unable to bind to the CD28 stem region on cells and could not inhibit CD28 shedding from the cell membrane as described above. At the cellular level, a standard sandwich ELISA was used to measure the levels of human sCD28 in the supernatant of HEK cells overexpressing human CD28 ( Fig.18 ), PHA and IL-2 activated isolated CD4 T cells ( Fig.19 ) and superantigen activated PBMC ( Fig. 20 ) to confirm the efficacy of the VHH clones in inhibiting sCD28 shedding. As expected, M9 Fab did not reduce sCD28 levels in the supernatant, further emphasizing the importance of the size and architecture of the blocker on its ability to actually block shedding.

[0304] Crucially, the VHH clones were found not to impair human CD28 functionality. Using flow cytometry, the VHH clones were found not to alter the magnitude of CD86 binding to membrane CD28 ( Fig.15 .). A standard sandwich ELISA was used to show that the VHH clones did not agonize CD28, as measured by secretion levels of the inflammatory cytokine interferon gamma ( Fig.16 ). The activating antibody CD28.2 was used as a positive control. Similarly, a standard sandwich ELISA was used to show that the VHH clones did not antagonize CD80-Fc stimulation via CD28, as measured by secretion levels of the cytokine IL-2 ( Fig.21 ).

[0305] Example 8: Design of other small reagents to inhibit sCD28 shedding from the cell surface

[0306] Fab fragment generation is carried out using commercial kits or commercial services. The CDR regions of antibody M9 are used for Fab generation because they have been shown to bind to suitable deisotopes. The efficacy of the obtained Fab fragments is first tested by binding assays with recombinant human CD28 and dimer stem region peptides to confirm that this binding is retained. The binding to the surface mCD28 of mouse cells and human immune cells expressing human CD28 is determined by FACS. Antibody CD28.2 is used as a positive control. Direct inhibition of sCD28 shedding is tested in immune cell cultures after stimulation. sCD28 in the culture medium is measured by sandwich ELISA when cells are present and absent Fab fragments. The effect of Fab fragments with shedding inhibition on CD28 signaling is determined. First, the agonism is tested by determining the ability of Fab fragments to induce secretion of proinflammatory cytokines (such as interferon gamma) from T cells. Secondly, the ability to block CD80-Fc (agonist) binding is used to test the anti-anti-antibody properties of Fab fragments.

[0307] Single chain antibody production using the M9 CDRs was performed by standard methods, using commercial services, or by inserting the CDRs into a scFV framework. Purification was performed and the resulting antibodies were evaluated by the same assays as described for the Fab fragments.

[0308] Single domain antibodies are generated by one of two strategies. 1) Naive library – Phage library using naive llama-derived VHHs – This library consists of VHH sequences taken from naive llama spleens, i.e., B cells are extracted and the entire available repertoire of VHH CDRs is sequenced. These CDRs are introduced into phage to generate the library. The library is screened against recombinant CD28 ectodomain and dimer stem region to find antibodies that specifically bind to sCD28. 2) Immune library – Llamas or other animal species or sharks are immunized with cells that overexpress CD28. Following cellular immunization, spleens are extracted and the available repertoire of VHH CDRs is sequenced. Hybridomas are made from the extracted spleen B cells. The resulting antibodies are introduced into phage to generate the library, and the library is screened against recombinant CD28 ectodomain and dimer stem region peptides to find antibodies that specifically bind to sCD28. Single domain antibodies with specific binding were evaluated for shedding blockade and agonism / antagonism as was done for Fab fragments and single chain antibodies.

[0309] Although the present invention has been described in conjunction with its specific embodiments, it is obvious that many substitutions, modifications and variations will be apparent to those skilled in the art. Therefore, it is intended to include all such substitutions, modifications and variations that fall within the spirit and broad scope of the appended claims. Sequence Listing <110> Biogen Biologics <120> Small shedding blocker <130> BDB-P-008-PCT <150> 62 / 954802 <151> December 30, 2019 <150> 62 / 942,240 <151> December 2, 2019 <150> 62 / 818351 <151> March 14, 2019 <160> 49 <170> PatentIn version 3.5 <210> 1 <211> 220 <212> PRT <213> Homo sapiens <400> 1 Met Leu Arg Leu Leu Leu Ala Leu Asn Leu Phe Pro Ser Ile Gln Val 1 5 10 15 Thr Gly Asn Lys Ile Leu Val Lys Gln Ser Pro Met Leu Val Ala Tyr 20 25 30 Asp Asn Ala Val Asn Leu Ser Cys Lys Tyr Ser Tyr Asn Leu Phe Ser 35 40 45 Arg Glu Phe Arg Ala Ser Leu His Lys Gly Leu Asp Ser Ala Val Glu 50 55 60 Val Cys Val Val Tyr Gly Asn Tyr Ser Gln Gln Leu Gln Val Tyr Ser 65 70 75 80 Lys Thr Gly Phe Asn Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr 85 90 95 Phe Tyr Leu Gln Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys 100 105 110 Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser 115 120 125 Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro 130 135 140 Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly 145 150 155 160 Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile 165 170 175 Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met 180 185 190 Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro 195 200 205 Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 210 215 220 <210> 2 <211> 202 <212> PRT <213> Homo sapiens <400> 2 Asn Lys Ile Leu Val Lys Gln Ser Pro Met Leu Val Ala Tyr Asp Asn 1 5 10 15 Ala Val Asn Leu Ser Cys Lys Tyr Ser Tyr Asn Leu Phe Ser Arg Glu 20 25 30 Phe Arg Ala Ser Leu His Lys Gly Leu Asp Ser Ala Val Glu Val Cys 35 40 45 Val Val Tyr Gly Asn Tyr Ser Gln Gln Leu Gln Val Tyr Ser Lys Thr 50 55 60 Gly Phe Asn Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr Phe Tyr 65 70 75 80 Leu Gln Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys Lys Ile 85 90 95 Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly 100 105 110 Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe 115 120 125 Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val 130 135 140 Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp 145 150 155 160 Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met 165 170 175 Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala 180 185 190 Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 195 200 <210> 3 <211> 663 <212> DNA <213> Homo sapiens <400> 3 atgctcaggc tgctcttggc tctcaactta ttcccttcaa ttcaagtaac aggaaacaag 60 attttggtga agcagtcgcc catgcttgta gcgtacgaca atgcggtcaa ccttagctgc 120 aagtattcct acaatctctt ctcaagggag ttccgggcat cccttcacaa aggactggat 180 agtgctgtgg aagtctgtgt tgtatatggg aattactccc agcagcttca ggtttactca 240 aaaacggggt tcaactgtga tgggaaattg ggcaatgaat cagtgacatt ctacctccag 300 aatttgtatg ttaaccaaac agatatttac ttctgcaaaa ttgaagttat gtatcctcct 360 ccttacctag acaatgagaa gagcaatgga accattatcc atgtgaaagg gaaacacctt 420 tgtccaagtc ccctatttcc cggaccttct aagccctttt gggtgctggt ggtggttggt 480 ggagtcctgg cttgctatag cttgctagta acagtggcct ttattatttt ctgggtgagg 540 agtaagagga gcaggctcct gcacagtgac tacatgaaca tgactccccg ccgccccggg 600 cccacccgca agcattacca gccctatgcc ccaccacgcg acttcgcagc ctatcgctcc 660 tga 663 <210> 4 <211> 27 <212> PRT <213> Homo sapiens <400> 4 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 20 25 <210> 5 <211> 139 <212> PRT <213> Homo sapiens <400> 5 Met Leu Arg Leu Leu Leu Ala Leu Asn Leu Phe Pro Ser Ile Gln Val 1 5 10 15 Thr Gly Asn Lys Ile Leu Val Lys Gln Ser Pro Met Leu Val Ala Tyr 20 25 30 Asp Asn Ala Val Asn Leu Ser Cys Lys Tyr Ser Tyr Asn Leu Phe Ser 35 40 45 Arg Glu Phe Arg Ala Ser Leu His Lys Gly Leu Asp Ser Ala Val Glu 50 55 60 Val Cys Val Val Tyr Gly Asn Tyr Ser Gln Gln Leu Gln Val Tyr Ser 65 70 75 80 Lys Thr Gly Phe Asn Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr 85 90 95 Phe Tyr Leu Gln Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys 100 105 110 Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser 115 120 125 Asn Gly Thr Ile Ile His Val Lys Gly Glu Glu 130 135 <210> 6 <211> 121 <212> PRT <213> Homo sapiens <400> 6 Asn Lys Ile Leu Val Lys Gln Ser Pro Met Leu Val Ala Tyr Asp Asn 1 5 10 15 Ala Val Asn Leu Ser Cys Lys Tyr Ser Tyr Asn Leu Phe Ser Arg Glu 20 25 30 Phe Arg Ala Ser Leu His Lys Gly Leu Asp Ser Ala Val Glu Val Cys 35 40 45 Val Val Tyr Gly Asn Tyr Ser Gln Gln Leu Gln Val Tyr Ser Lys Thr 50 55 60 Gly Phe Asn Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr Phe Tyr 65 70 75 80 Leu Gln Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys Lys Ile 85 90 95 Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly 100 105 110 Thr Ile Ile His Val Lys Gly Glu Glu 115 120 <210> 7 <211> 538 <212> DNA <213> Homo sapiens <400> 7 atgctcaggc tgctcttggc tctcaactta ttcccttcaa ttcaagtaac aggaaacaag 60 attttggtga agcagtcgcc catgcttgta gcgtacgaca atgcggtcaa ccttagctgc 120 aagtattcct acaatctctt ctcaagggag ttccgggcat cccttcacaa aggactggat 180 agtgctgtgg aagtctgtgt tgtatatggg aattactccc agcagcttca ggtttactca 240 aaaacggggt tcaactgtga tgggaaattg ggcaatgaat cagtgacatt ctacctccag 300 aatttgtatg ttaaccaaac agatatttac ttctgcaaaa ttgaagttat gtatcctcct 360 ccttacctag acaatgagaa gagcaatgga accattatcc atgtgaaagg tgaggagtaa 420 gaggagcagg ctcctgcaca gtgactacat gaacatgact ccccgccgcc ccgggcccac 480 ccgcaagcat taccagccct atgccccacc acgcgacttc gcagcctatc gctcctga 538 <210> 8 <211> 110 <212> PRT <213> Homo sapiens <400> 8 Met Leu Val Ala Tyr Asp Asn Ala Val Asn Leu Ser Cys Lys Tyr Ser 1 5 10 15 Tyr Asn Leu Phe Ser Arg Glu Phe Arg Ala Ser Leu His Lys Gly Leu 20 25 30 Asp Ser Ala Val Glu Val Cys Val Val Tyr Gly Asn Tyr Ser Gln Gln 35 40 45 Leu Gln Val Tyr Ser Lys Thr Gly Phe Asn Cys Asp Gly Lys Leu Gly 50 55 60 Asn Glu Ser Val Thr Phe Tyr Leu Gln Asn Leu Tyr Val Asn Gln Thr 65 70 75 80 Asp Ile Tyr Phe Cys Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu 85 90 95 Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile His Val Lys Gly 100 105 110 <210> 9 <211> 16 <212> PRT <213> Homo sapiens <400> 9 Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro 1 5 10 15 <210> 10 <211> 19 <212> PRT <213> Homo sapiens <400> 10 His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro 1 5 10 15 Ser Lys Pro <210> 11 <211> 7 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 11 Gly Tyr Thr Leu Thr Asn Tyr 1 5 <210> 12 <211> 6 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 12 Asn Thr Tyr Thr Gly Lys 1 5 <210> 13 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 13 Gly Asp Ala Asn Gln Gln Phe Ala Tyr 1 5 <210> 14 <211> 11 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 14 Lys Ala Ser Gln Asp Ile Asn Ser Tyr Leu Ser 1 5 10 <210> 15 <211> 7 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 15 Arg Ala Asn Arg Leu Val Asp 1 5 <210> 16 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 16 Leu Gln Tyr Asp Glu Phe Pro Pro Thr 1 5 <210> 17 <211> 10 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 17 Gly Phe Thr Phe Ser Ser Tyr Tyr Met Ser 1 5 10 <210> 18 <211> 17 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 18 Thr Ile Ser Asp Gly Gly Asp Asn Thr Tyr Tyr Ala Gly Thr Val Thr 1 5 10 15 Gly <210> 19 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 19 Ile His Trp Pro Tyr Tyr Phe Asp Ser 1 5 <210> 20 <211> 10 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 20 Arg Ala Ser Ser Ser Val Ser Tyr Met Asn 1 5 10 <210> twenty one <211> 7 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> twenty one Ala Thr Ser Asp Leu Ala Ser 1 5 <210> twenty two <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> twenty two Gln Gln Trp Ser Ser His Pro Pro Thr 1 5 <210> 23 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 23 Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Leu Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Tyr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Asp Gly Gly Asp Asn Thr Tyr Tyr Ala Gly Thr Val 50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Asp Phe Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile His Trp Pro Tyr Tyr Phe Asp Ser Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser 115 <210> 24 <211> 354 <212> DNA <213> Artificial sequence <220> <223> Synthesis <400> 24 gacgtgaagc tcgtggagtc tgggggaggc ttagtgaagc ttggagggtc cctgaaactc 60 tcctgtgtag cctctggatt cactttcagt agctattaca tgtcttgggt tcgccagact 120 ccggagaaga ggctggagatg ggtcgcgacc ataagtgatg gtggtgataa cacctactac 180 gcaggcactg tgacgggccg attcaccatc tccagagact ttgccaagaa caccctgtac 240 ctgcaaatga acagtctgac ctctgaggac acagccgtgt attactgtgc aagaattcat 300 tggccttact attttgactc ctggggccaa ggcaccactc tcacagtctc ctca 354 <210> 25 <211> 106 <212> PRT <213> Artificial sequence <220> <223> Synthesis <400> 25 Gln Phe Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly 1 5 10 15 Glu Met Leu Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Asn Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Ala Thr Ser Asp Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser His Pro Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Arg 100 105 <210> 26 <211> 318 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 26 caatttgttc tctcccagtc tccagcaatc ctgtctgcat ctcccgggga gatgctcaca 60 atgacttgca gggccagctc aagtgtaagt tatatgaact ggtatcagca gaagccagga 120 tcttccccca aaccctggat ttatgccaca tccgacctgg cttctggagt ccctgctcgc 180 ttcagtggca gtgggtctgg gacctcttat tctctcacaa tcagcagagt ggaggctgaa 240 gatgctgcca cttattactg ccagcagtgg agtagtcacc cacccacgtt cggagggggg 300 accaagctgg aaataaga 318 <210> 27 <211> 15 <212> PRT <213> Homo sapiens <400> 27 Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser 1 5 10 15 <210> 28 <211> 134 <212> PRT <213> Homo sapiens <400> 28 Asn Lys Ile Leu Val Lys Gln Ser Pro Met Leu Val Ala Tyr Asp Asn 1 5 10 15 Ala Val Asn Leu Ser Cys Lys Tyr Ser Tyr Asn Leu Phe Ser Arg Glu 20 25 30 Phe Arg Ala Ser Leu His Lys Gly Leu Asp Ser Ala Val Glu Val Cys 35 40 45 Val Val Tyr Gly Asn Tyr Ser Gln Gln Leu Gln Val Tyr Ser Lys Thr 50 55 60 Gly Phe Asn Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr Phe Tyr 65 70 75 80 Leu Gln Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys Lys Ile 85 90 95 Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly 100 105 110 Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe 115 120 125 Pro Gly Pro See Light Pro 130 <210> 29 <211> 17 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 29 Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro 1 5 10 15 Light <210> 30 <211> 117 <212> PRT <213> artificial <220> <223> synthesis <400> 30 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Ala Ser Ile Asn 20 25 30 Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Ser Gln Arg Glu Leu Val 35 40 45 Ala Ala Ile Ser Gly Gly Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Val 85 90 95 Val Asp Leu Tyr Gly Ser Asp Tyr Trp Asp Trp Gly Gln Gly Thr Gln 100 105 110 Val Thr Val Ser Ser 115 <210> 31 <211> 117 <212> PRT <213> artificial <220> <223> synthesis <400> 31 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Leu Phe Ser Ile Asn 20 25 30 Ala Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Ala Ile Thr Ser Ser Gly Ser Thr Asn Tyr Ala Asn Ser Val Lys 50 55 60 Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Val 85 90 95 Val Asp Glu Tyr Gly Ser Asp Tyr Trp Ile Trp Gly Gln Gly Thr Gln 100 105 110 Val Thr Val Ser Ser 115 <210> 32 <211> 117 <212> PRT <213> artificial <220> <223> synthesis <400> 32 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ile Asn 20 25 30 Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Arg Val 35 40 45 Ala Ala Ile Thr Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Asn Leu Glu Pro Arg Asp Ala Gly Val Tyr Tyr Cys Val 85 90 95 Val Asp Leu Tyr Gly Glu Asp Tyr Trp Ile Trp Gly Gln Gly Thr Gln 100 105 110 Val Thr Val Ser Ser 115 <210> 33 <211> 5 <212> PRT <213> Artificial <220> <223> synthesis <400> 33 I'm Alas, I'm Gly 1 5 <210> 34 <211> 16 <212> PRT <213> Artificial <220> <223> synthesis <400> 34 Ala Ile Ser Gly Gly Gly Asp Thr Tyr Tyr Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 35 <211> 9 <212> PRT <213> Artificial <220> <223> synthesis <400> 35 Asp Leu Tyr Gly Ser Asp Tyr Trp Asp 1 5 <210> 36 <211> 5 <212> PRT <213> Artificial <220> <223> synthesis <400> 36 I'm Ala Met Ala 1 5 <210> 37 <211> 16 <212> PRT <213> Artificial <220> <223> synthesis <400> 37 Ala Ile Thr Ser Ser Gly Ser Thr Asn Tyr Ala Asn Ser Val Lys Gly 1 5 10 15 <210> 38 <211> 9 <212> PRT <213> Artificial <220> <223> synthesis <400> 38 Asp Glu Tyr Gly Ser Asp Tyr Trp Ile 1 5 <210> 39 <211> 16 <212> PRT <213> Artificial <220> <223> synthesis <400> 39 Ala Ile Thr Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 40 <211> 9 <212> PRT <213> Artificial <220> <223> synthesis <400> 40 Asp Leu Tyr Gly Glu Asp Tyr Trp Ile 1 5 <210> 41 <211> 5 <212> PRT <213> Artificial <220> <223> synthesis <220> <221> X <222> (5) <223> X is G or A <400> 41 I'm a bitch 1 5 <210> 42 <211> 16 <212> PRT <213> Artificial <220> <223> synthesis <220> <221> X <222> (3) <223> X is S or T <220> <221> X <222> (4) <223> X is G or S <220> <221> X <222> (5) <223> X is G or S <220> <221> X <222> (7) <223> X is D or S <220> <221> X <222> (9)..(9) <223> X is Y or N <220> <221> X <222> (12)..(12) <223> X is D or N <400> 42 Ala Ile Xaa Xaa Xaa Gly Xaa Thr Xaa Tyr Ala Xaa Ser Val Lys Gly 1 5 10 15 <210> 43 <211> 9 <212> PRT <213> Artificial <220> <223> synthesis <220> <221> X <222> (2) <223> X is E or L <220> <221> X <222> (5) <223> X is E or S <220> <221> X <222> (9)..(9) <223> X is D or I <400> 43 Asp Xaa Tyr Gly Xaa Asp Tyr Trp Xaa 1 5 <210> 44 <211> 9 <212> PRT <213> Artificial <220> <223> synthesis <220> <221> X <222> (2) <223> X is E or L <220> <221> X <222> (9)..(9) <223> X is D or I <400> 44 Asp Xaa Tyr Gly Ser Asp Tyr Trp Xaa 1 5 <210> 45 <211> 126 <212> PRT <213> Artificial <220> <223> synthesis <400> 45 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Ala Ser Ile Asn 20 25 30 Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Ser Gln Arg Glu Leu Val 35 40 45 Ala Ala Ile Ser Gly Gly Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Val 85 90 95 Val Asp Leu Tyr Gly Ser Asp Tyr Trp Asp Trp Gly Gln Gly Thr Gln 100 105 110 Val Thr Val Ser Ser Ala Ala Ala His His His His His His 115 120 125 <210> 46 <211> 126 <212> PRT <213> Artificial <220> <223> Synthetic <400> 46 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Leu Phe Ser Ile Asn 20 25 30 Ala Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Ala Ile Thr Ser Ser Gly Ser Thr Asn Tyr Ala Asn Ser Val Lys 50 55 60 Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Val 85 90 95 Val Asp Glu Tyr Gly Ser Asp Tyr Trp Ile Trp Gly Gln Gly Thr Gln 100 105 110 Val Thr Val Ser Ser Ala Ala Ala His His His His His His 115 120 125 <210> 47 <211> 126 <212> PRT <213> Artificial <220> <223> Synthetic <400> 47 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ile Asn 20 25 30 Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Arg Val 35 40 45 Ala Ala Ile Thr Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Asn Leu Glu Pro Arg Asp Ala Gly Val Tyr Tyr Cys Val 85 90 95 Val Asp Leu Tyr Gly Glu Asp Tyr Trp Ile Trp Gly Gln Gly Thr Gln 100 105 110 Val Thr Val Ser Ser Ala Ala Ala His His His His His His 115 120 125 <210> 48 <211> 145 <212> PRT <213> Homo sapiens <400> 48 Met Leu Arg Leu Leu Leu Ala Leu Asn Leu Phe Pro Ser Ile Gln Val 1 5 10 15 Thr Gly Asn Lys Ile Leu Val Lys Gln Ser Pro Met Leu Val Ala Tyr 20 25 30 Asp Asn Ala Val Asn Leu Ser Cys Lys Tyr Ser Tyr Asn Leu Phe Ser 35 40 45 Arg Glu Phe Arg Ala Ser Leu His Lys Gly Leu Asp Ser Ala Val Glu 50 55 60 Val Cys Val Val Tyr Gly Asn Tyr Ser Gln Gln Leu Gln Val Tyr Ser 65 70 75 80 Lys Thr Gly Phe Asn Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr 85 90 95 Phe Tyr Leu Gln Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys 100 105 110 Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser 115 120 125 Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro 130 135 140 Leu 145 <210> 49 <211> 127 <212> PRT <213> Homo sapiens <400> 49 Asn Lys Ile Leu Val Lys Gln Ser Pro Met Leu Val Ala Tyr Asp Asn 1 5 10 15 Ala Val Asn Leu Ser Cys Lys Tyr Ser Tyr Asn Leu Phe Ser Arg Glu 20 25 30 Phe Arg Ala Ser Leu His Lys Gly Leu Asp Ser Ala Val Glu Val Cys 35 40 45 Val Val Tyr Gly Asn Tyr Ser Gln Gln Leu Gln Val Tyr Ser Lys Thr 50 55 60 Gly Phe Asn Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr Phe Tyr 65 70 75 80 Leu Gln Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys Lys Ile 85 90 95 Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly 100 105 110 Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu 115 120 125

Claims

1. A single domain antibody that binds to membrane CD28 (mCD28) on a cell surface and inhibits protease cleavage of said mCD28, comprising three CDRs, wherein: CDR1 consists of the amino acid sequence set forth in SEQ ID NO:33 (INAMG), CDR2 consists of the amino acid sequence set forth in SEQ ID NO:34 (AISGGGDTYYADSVKG), and CDR3 consists of the amino acid sequence set forth in SEQ ID NO:35 (DLYGSDYWD); CDR1 consists of the amino acid sequence set forth in SEQ ID NO:36 (INAMA), CDR2 consists of the amino acid sequence set forth in SEQ ID NO:37 (AITSSGSTNYANSVKG), and CDR3 consists of the amino acid sequence set forth in SEQ ID NO:38 (DEYGSDYWI); or CDR1 consists of the amino acid sequence described in SEQ ID NO:33 (INAMG), CDR2 consists of the amino acid sequence described in SEQ ID NO:39 (AITSGGSTNYADSVKG), and CDR3 consists of the amino acid sequence described in SEQ ID NO:40 (DLYGEDYWI).

2. The single domain antibody of claim 1, wherein the single domain antibody is a camelid antibody or a shark antibody.

3. The single domain antibody of claim 2, wherein the single domain antibody is a camelid antibody.

4. The single domain antibody according to claim 3, wherein the camelid antibody consists of a sequence selected from the group consisting of: a.EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVA AISGGGDTYYADSVKGRFTISRDDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGS DYWDWGQGTQVTVSS (SEQ ID NO: 30); and c. QVQLVESGGGLVQAGGSLRLSCAASGSIFSINAMGWYRQAPGKQRERVA AITSGGSTNYADSVKGRFTISSRDNAKNTVYLQMNNLEPRDAGVYYCVVDLYGE DYWIWGQGTQVTVSS (SEQ ID NO: 32).

5. The single domain antibody according to any one of claims 1 to 3, wherein the single domain antibody is humanized.

6. The single domain antibody according to any one of claims 1 to 3, wherein the single domain antibody is not a CD28 agonist.

7. The single domain antibody according to any one of claims 1 to 3, wherein the single domain antibody is not a CD28 antagonist.

8. The single domain antibody according to any one of claims 1 to 3, wherein the single domain antibody neither degrades the mCD28 nor inhibits mCD28-mediated immune cell activation.

9. The single domain antibody according to any one of claims 1 to 3, wherein the antigen binding fragment of the antibody does not induce antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

10. The single domain antibody according to any one of claims 1 to 3, wherein the single domain antibody binds within the stalk region of CD28.

11. The single domain antibody according to claim 10, wherein the stem region comprises the amino acid sequence GKHLCPSPLFPGPSKP (SEQ ID NO: 9) or KGKHLCPSPLFPGPS (SEQ ID NO: 27).

12. The single domain antibody according to claim 11, wherein the stem region consists of the amino acid sequence HVKGKHLCPSPLFPGPSKP (SEQ ID NO: 10).

13. The single domain antibody according to any one of claims 1 to 3, wherein said single domain antibody binds at least one protease at the cleavage site.

14. The single domain antibody according to any one of claims 1 to 3, wherein said single domain antibody is inhibited from protease cleavage by at least one protease.

15. The single domain antibody according to claim 13, wherein the at least one protease is at least one metalloprotease.

16. The single domain antibody of claim 15, wherein the at least one metalloprotease is MMP-2, MMP-13 or a combination thereof.

17. Use of a single domain antibody according to any one of claims 1 to 3 in the manufacture of a medicament for treating and / or preventing cancer in a subject in need thereof, wherein the cancer is selected from melanoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, renal cancer, gastric cancer, and colorectal cancer.

18. Use of a single domain antibody according to any one of claims 1 to 3 in the manufacture of a medicament for improving PD-1 and / or PD-L1 based immunotherapy in a subject suffering from cancer, wherein the cancer is selected from melanoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, renal cancer, gastric cancer, and colorectal cancer.

19. The use according to claim 17, wherein the cancer is selected from melanoma, head and neck cancer, non-small cell lung cancer, ovarian cancer and colorectal cancer.

20. The use according to claim 18, wherein the cancer is selected from melanoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, and colorectal cancer.

21. A pharmaceutical composition comprising the single domain antibody according to any one of claims 1 to 3, and a pharmaceutically acceptable carrier, excipient or adjuvant.

22. Use of a pharmaceutical composition according to claim 21 in the manufacture of a medicament for treating and / or preventing cancer or improving PD-1 and / or PD-L1-based immunotherapy in a subject suffering from cancer, wherein the cancer is selected from melanoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, renal cancer, gastric cancer, and colorectal cancer.

23. The use according to claim 22, wherein the cancer is selected from melanoma, head and neck cancer, non-small cell lung cancer, ovarian cancer and colorectal cancer.

24. A kit comprising the single domain antibody according to any one of claims 1 to 3 and at least one of the following: a. PD-1 and / or PD-L1 based immunotherapeutics; and b. A tag describing the single domain antibody according to any one of claims 1 to 3 for use with the PD-1 and / or PD-L1 based immunotherapeutic agent.

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