Low molecular weight release inhibitor

Abstract

An agent that is less than 100 kilodaltons in size, binds to a membrane immunoreceptor on the surface of a cell, and inhibits proteolytic cleavage of the immunoreceptor is provided. Also provided are methods for treating cancer and improving immunotherapy that include administering the agent.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 954,802, filed December 30, 2019, U.S. Provisional Patent Application No. 62 / 942,240, filed December 2, 2019, and U.S. Provisional Patent Application No. 62 / 818,351, filed March 14, 2019, the contents of all of which are incorporated herein by reference in their entireties.

[0002] The present invention is in the field of immunomodulation and immunotherapy. [Background technology]

[0003] The adaptive immune system plays a key role in regulating and protecting against pathogens and cancer cells, primarily by coordinating the stimulation of antigen-specific helper CD4+ and cytotoxic CD8+ T cells. Durable and sustained activation of T cells by antigen-presenting cells (APCs) involves i) engagement of the T cell receptor (TCR) with peptides presented by the major histocompatibility complex (MHC) on the APC, and ii) the costimulatory CD28 receptor on the T cell, which binds to B7-1 (CD80) and B7-2 (CD86) ligands, which are also expressed by APCs. The biological consequences of CD28 costimulation are numerous and include T cell cycle control, expansion, differentiation, and amplification of TCR stimulation by lowering the threshold required to achieve immune effector function.

[0004] In contrast to the activating costimulatory molecule CD28, its structural homolog, cytotoxic T-lymphocyte-associated 4 (CTLA-4), is an inhibitory costimulatory receptor with membrane expression driven by CD28 triggering. Both CTLA-4 and CD28 are type I transmembrane proteins. Their extracellular portions consist of a single V-set immunoglobulin superfamily (Ig-V) domain homocovalently linked by a cysteine residue located outside the IgV domain proximal to the transmembrane region. CTLA-4 and CD28, although similar, differ in affinity and quaternary structure configuration. CTLA-4 has been found to have a higher binding affinity for B7 molecules, a different dimerization mode than CD28, and a different stoichiometry of binding with covalent ligands. CD28 exhibits monovalent binding stoichiometry, while CTLA-4 interacts in a bivalent manner. Thus, CTLA-4 binds to B7 molecules with much higher affinity and avidity than CD28, thereby down-regulating T cell responses and favoring the initiation of antigen-specific tolerance.

[0005] Several costimulatory molecules have been shown to have several physiological forms. In addition to membrane-bound forms, soluble forms expressed in naive immune cells have also been reported, adding to the complexity of T cell biology. The soluble form of CD28 (sCD28) is believed to result from an alternatively spliced gene product. The splicing event results in a frameshift, adding two glutamic acid residues after glycine 137 before translation is terminated. The final product lacks the entire transmembrane and cytoplasmic domains and, importantly, the cysteine residue at position 141 that mediates the disulfide bond in dimeric CD28 (Magistrelli G., Biochem Biophys Res Commun, 1999). The biological function and counter-receptor binding of the soluble form of monomeric CD28 have been investigated (Hebbar, M., Clin Exp Immunol, 2004), and it has been shown to inhibit T cell proliferation. However, dimeric sCD28 has been suggested to have a regulatory role in suppressing T cell function by binding to B7 molecules (Sun, Z., Centr Eur J Immunol, 2014; Hebbar, M., Clin Exp Immunol, 2004). Surprisingly, elevated levels of sCD28 molecules have been reported in the serum of patients with autoimmune diseases (Wong, CK, Rheumatol, 2005; Hamzaoui, K., Clin Exp Rheumatol, 2005; Hebbar, M., Clin Exp Immunol, 2004; Sun, Z., Clin Immunol, 2014). The exact source of sCD28 remains a topic of debate. Using an in vitro model of T cell activation that reflects the persistent inflammatory state of T cells in autoimmune patients, it has been shown that during the process of T cell activation, transcription of the alternative soluble form is suppressed and only the full-length membrane form of CD28 is evident, while the amount of sCD28 increases in culture (Hebbar, M., Clin Exp Immunol, 2004). This phenomenon has led to the suggestion that active shedding of the membrane form of CD28 is responsible for the rise in the soluble molecule in serum, but this has not yet been proven.Active shedding during T cell activation has previously been described as a regulatory mechanism to counter sustained activation by proteolytic degradation of adhesion molecules.

[0006] While CTLA-4 limits the amplitude of early T cell responses, another inhibitory receptor, PD-1, suppresses peripheral T cell function. PD-1 expression is elevated during T cell activation, and its known ligands are the B7 family homologs: B7-H1 (PD-L1) and B7-H2 (PD-L2). These homologs are found on APCs and cancer cells, driving activated T cells into a state of cellular anergy and dampening the immune response. Therefore, targeted therapies against CTLA-4 and the PD-1 / PD-L1 axis have shown clinical activity in various types of cancer. Recent studies have demonstrated that the CD28 signaling pathway is targeted and suppressed by PD-1 (Hui, E., Science, 2017), and simultaneously demonstrated that an intact and active CD28 / B7 axis is essential for effective PD-1 therapy (Kamphorst, A.O., Science, 2017). Summary of the Invention [Problem to be solved by the invention]

[0007] However, not all patients respond to PD-1-based immunotherapy or immunotherapy in general, and relapse often occurs. Therefore, there is a great need for methods and molecules that can improve the ability of patients' immune cells to attack cancer. [Means for solving the problem]

[0008] The present invention provides agents of less than 100 kilodaltons that bind to membrane CD28 (mCD28) on the cell surface and inhibit the proteolytic cleavage of mCD28. Also provided are methods for treating and preventing cancer, and methods for improving PD-1 / PD-L1-based immunotherapy, that include administering the agents.

[0009] According to a first aspect, there is provided an agent that binds to membrane CD28 (mCD28) on the surface of a cell and inhibits proteolytic cleavage of mCD28, the agent being less than 100 kilodaltons (kDa).

[0010] According to another aspect, there is provided a method of reducing soluble CD28 (sCD28) levels in a subject in need thereof, the method comprising administering an agent of the invention.

[0011] 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.

[0012] According to another aspect, there is provided a method of improving PD-1 and / or PD-L1 based immunotherapy in a subject in need thereof, the method comprising administering an agent of the invention.

[0013] According to another aspect, there is provided a method for producing an agent that inhibits proteolytic cleavage of mCD28 on the cell surface, the method comprising: a. Obtaining an agent that binds to the CD28 extracellular domain or a fragment thereof, wherein the agent is less than 100 kDa; b. Testing the binding of the obtained agent to mCD28 on the cell surface; and c. selecting an agent that binds to cell surface mCD28; and d. Culturing host cells containing one or more vectors containing a nucleic acid sequence encoding an agent; and wherein the nucleic acid sequence comprises at least one of: i. Obtaining an agent that binds to the CD28 extracellular domain or a fragment thereof, wherein the agent is less than 100 kDa; ii. Testing the binding of the obtained agent to mCD28 on the cell surface; and iii. The nucleic acid sequence of an agent selected by selecting an agent that binds to cell surface mCD28; Thereby, an agent is generated that inhibits the proteolytic cleavage of mCD28 on the cell surface.

[0014] According to another aspect, there is provided a pharmaceutical agent produced by the method of the invention.

[0015] According to another aspect, a pharmaceutical composition is provided, comprising an agent of the invention and a pharmaceutically acceptable carrier, excipient or adjuvant.

[0016] According to another aspect, there is provided 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, the method comprising administering a pharmaceutical composition of the invention.

[0017] According to another aspect, there is provided a kit comprising at least one agent of the invention.

[0018] According to some embodiments, the agent is selected from an antigen-binding fragment of an antibody that specifically binds to CD28, a Fab fragment, a single chain antibody, a single domain antibody, a small molecule, and a peptide.

[0019] According to some embodiments, the agent is less than 50 kDa.

[0020] According to some embodiments, the single domain antibody is a camelid antibody or a shark antibody.

[0021] According to some embodiments, the camelid antibody comprises three CDRs: CDR1 comprises the amino acid sequence shown in SEQ ID NO: 33 (INAMG), CDR2 comprises the amino acid sequence shown in SEQ ID NO: 34 (AISGGGDTYYADSVKG), and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 35 (DLYGSDYWD), or 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 CDR1 comprises the amino acid sequence shown in SEQ ID NO: 33 (INAMG), CDR2 comprises the amino acid sequence shown in SEQ ID NO: 39 (AITSGGSTNYADSVKG), and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 40 (DLYGEDYWI).

[0022] According to some embodiments, the camelid antibody comprises: a.EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVTVSS (SEQ ID NO: 30), b. EVQLVESGGGLVQAGGSLRLSCAASGSLFSINAMAWYRQAPGKQRELVAAITSSGSTNYANSVKGRFTVSRDNAKNTMYLQMNSLKPEDTAVYYCVVDEYGSDYWIWGQGTQVTVSS (SEQ ID NO: 31), and c. QVQLVESGGGLVQAGGSLRLSCAASGSIFSINAMGWYRQAPGKQRERVAAITSGGSTNYADSVKGRFTISRDNAKNTVYLQMNNLEPRDAGVYYCVVDLYGEDYWIWGQGTQVTVSS (SEQ ID NO: 32).

[0023] According to some embodiments, the agent comprises three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), CDR-H1 comprises the amino acid sequence shown in SEQ ID NO: 17 (GFTFSSYYMS), CDR-H2 comprises the amino acid sequence shown in SEQ ID NO: 18 (TISDGGDNTYYAGTVTG), CDR-H3 comprises the amino acid sequence shown in SEQ ID NO: 19 (IHWPYYFDS), CDR-L1 comprises the amino acid sequence shown in SEQ ID NO: 20 (RASSSVSYMN), CDR-L2 comprises the amino acid sequence shown in SEQ ID NO: 21 (ATSDLAS), and CDR-L3 comprises the amino acid sequence shown in SEQ ID NO: 22 (QQWSSHPPT).

[0024] According to some embodiments, the agent is humanized.

[0025] According to some embodiments, the agent is not a CD28 agonist.

[0026] According to some embodiments, the agent is not a CD28 antagonist.

[0027] According to some embodiments, the agent does not degrade mCD28 or inhibit mCD28-mediated immune cell activation.

[0028] According to some embodiments, the antigen-binding fragment of an antibody does not induce antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0029] According to some embodiments, the agent binds within the stalk region of CD28.

[0030] According to some embodiments, the stalk region comprises the amino acid sequence GKHLCPSPLFPGPSKP (SEQ ID NO: 9) or KGKHLCPSPLFPGPS (SEQ ID NO: 27).

[0031] According to some embodiments, the stalk region consists of the amino acid sequence HVKGKHLCPSPLFPGPSKP (SEQ ID NO: 10).

[0032] According to some embodiments, the agent binds to a cleavage site for at least one protease.

[0033] According to some embodiments, the agent inhibits proteolytic cleavage by at least one protease.

[0034] According to some embodiments, the at least one protease is at least one metalloprotease.

[0035] According to some embodiments, the at least one metalloprotease is MMP-2, MMP-13, or a combination thereof.

[0036] According to some embodiments, the subject is suffering from cancer.

[0037] 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.

[0038] According to some embodiments, the cancer is selected from melanoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, and colorectal cancer.

[0039] According to some embodiments, the method does not degrade mCD28 or reduce mCD28-mediated immune cell activation.

[0040] According to some embodiments, the subject's blood prior to administration contains at least 5 ng / ml of sCD28.

[0041] According to some embodiments, obtaining comprises obtaining an agent less than 50 kDa, and the obtained agent is less than 50 kDa.

[0042] According to some embodiments, the method further comprises testing the ability of the agent to block protease cleavage of mCD28 on the cell surface.

[0043] According to some embodiments, the protease is selected from MMP-2 and MMP-13.

[0044] According to some embodiments, obtaining the agent comprises: a. Immunizing a shark or camelid with the CD28 extracellular domain or a fragment thereof and collecting antibodies from the immunized organism; and b. screening a library of drugs for binding to the CD28 extracellular domain or a fragment thereof and selecting drugs that bind; It includes at least one of the following:

[0045] According to some embodiments, the CD28 extracellular domain or fragment thereof is a dimer or a monomer.

[0046] According to some embodiments, a. collecting antibodies comprises extracting B cells from the spleen of an immunized shark or camelid; or b. Selecting the agents that bind includes sequencing the selected agents and generating a recombinant form of the agent from the sequence.

[0047] According to some embodiments, the method further comprises assaying mCD28 downstream signaling in the presence of the obtained agents and selecting at least one agent that neither substantially agonizes nor substantially antagonizes mCD28 signaling.

[0048] According to some embodiments, the kit comprises: a. anti-PD-1 and / or PD-L1 immunotherapy, and b. A label indicating that the agent of the invention is for use in conjunction with PD-1 and / or PD-L1 based immunotherapy; The method further includes at least one of:

[0049] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0050] [Figure 1] Soluble CD28 is generated during PBMC stimulation and neutralized by the addition of protease inhibitors (PIs). Figure 1 shows a bar graph (top panel) 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), as quantified by human CD28 ELISA. A cocktail of protease inhibitors was added at the indicated concentrations. Overall health and effector activity were examined by interferon-γ secretion (bottom panel). [Figure 2] During T cell stimulation with PHA, soluble CD28 is generated and neutralized by the addition of protease inhibitors. Bar graphs show Jurkat cells (top left) or isolated human CD4 T cells (top right) stimulated with increasing concentrations of PHA (1–4 μg / mL, top graph) in the presence of a constant concentration of protease inhibitor cocktail (2 μM). In a separate setup, Jurkat T cells (1 μg / mL PHA, bottom left) or human CD4 T cells (2 μg / mL PHA, bottom right) were stimulated with a fixed PHA concentration and an adjusted concentration of protease inhibitor cocktail (0.5–2 μM). The concentration of human CD28 in the supernatant was quantified by a standardized sandwich ELISA. [Figure 3A-B]Specific ADAM-10 and ADAM-17 inhibitors eliminate the accumulation of soluble CD28 during activation of human PBMCs with SEB but do not interfere with their viability. (3A-B) Bar graphs of human PBMCs stimulated with SEB (1 ng / mL) in the presence of various concentrations (0.01-1 μM) of (3A) the ADAM-10-specific inhibitor (GI254023X) and (3B) the ADAM-17-specific inhibitor (TMI-1). Cell viability in the different treatments was assessed using an MTT assay (top panel). The concentration of human CD28 in the supernatant (bottom panel) was quantified by standardized sandwich ELISA. [Figure 4A-D] Soluble CD28 is generated during PBMC stimulation. (4A) Bar graph of immature dendritic cells mixed at a 1:5 ratio with CD3 T cells from the same donor without CMV (black bars) or with CMV peptide (dark gray bars). Controls for each cell population, either alone or with CMV, are shown in light gray bars. The concentration of human CD28 in the supernatant was quantified by a standardized sandwich ELISA. (Figures 4B-4D) Bar graphs of human PBMCs stimulated for 24 hours with (4B) CMV, (4C) SEB, or (4D) SEB in the presence of ADAM-10 and ADAM-17 inhibitors, and then transferred to clean cultures. The measurements in Figure 4D are taken 120 hours after cell transfer. [Figure 5] Soluble CD28 inhibits the secretion of effector cytokines. Bar graphs of human PBMCs stimulated with CMV (0.5 μg / mL) without (black bars) or with (gray bars) recombinant human CD28 at the indicated concentrations. Naive samples without CMV stimulation are shown in light gray bars. Human IFN-gamma concentrations in the supernatants were quantified by a standardized sandwich ELISA (Biolegend). [Figure 6]Soluble CD28 increases IL-6 cytokine secretion. Bar graphs of human PBMCs stimulated with CMV (0.5 μg / mL) without (black bars) or with (gray bars) recombinant human soluble CD28 at the indicated concentrations. Naive samples without CMV stimulation are shown in light gray bars. Human IL-6 concentrations in the supernatants were quantified by a standardized sandwich ELISA (Biolegend). [Figures 7A-E] (7A) Line graph of human PBMCs stimulated with CMV (0.5 μg / mL) in the presence of recombinant human soluble CD28 (gray triangles) or recombinant human soluble CTLA-4 (black circles) at the indicated concentrations. Supernatant concentrations of human IL-6, IFNγ, and IL-4 were quantified by standardized sandwich ELISA (Biolegend). Supernatant concentrations of human IL-8, IL-12p(40), and IL-10 were quantified by multiplex analysis using the Magpix system (Millipore). (7B) Bar graph of cytokine secretion from autologous monocytes and CD3+ MLRs. Naive samples without CMV stimulation are shown as light gray bars. CMV alone or IgG control are shown as black bars. Increasing concentrations of sCD28 are shown as dark gray bars. (7C) Line graph of lymphocyte clustering by human PBMCs stimulated with SEB (gray circles) or control IgG (gray triangles) in the presence of recombinant human soluble CD28. (7D) Bar graph of IDO secretion into culture measured by kynurenine ELISA kit from monocytes treated with or without recombinant human sCD28. (7E) Scatter plot of intracellular FACS of IDO in monocytes treated with or without recombinant human sCD28. [Figure 8A-C]Soluble CD28 inhibits anti-PD1 treatment. (8A) Bar graphs of human PBMCs stimulated for 3 days with SEB (200 ng / mL, left graph) or CMV peptide (0.5 μg / mL, right graph) 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 (dotted bars). (8B) Bar graphs of cytokine secretion from a monocyte MLR setup (naive - open bars, CMV only - light gray bars, sCD28 - black bars, MK-3475 - dark gray, sCD28 + MK-3475 - grid bars). Concentrations of human IFNγ, TGFβ, and IL-2 in the supernatants were quantified by standardized sandwich ELISA (Biolegend). (8C) Histograms of surface PD-L1 (left) and PD-L2 (right) expression on monocytes after incubation with control and sCD28. [Figure 9A-C] 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 with high soluble CD28 content were investigated repeatedly at several dilution factors. The concentration of human CD28 in the supernatant was quantified by a standardized sandwich ELISA internally corrected to correspond to the readings from human plasma samples. (9B) Bar graphs of INFγ secretion measured by sandwich ELISA from SEB-stimulated PBMCs from cancer patients (a sarcoma patient—top left, a kidney cancer patient—top right, and two different head and neck cancer patients—bottom) in the presence of sCD28, MK-3475, and combinations of the two. (9C) Bar graphs of the viability and proliferation of cancer cells SCC-25 alone, in combination with IL-6, in coculture with monocytes, or in coculture with monocytes and sCD28. [Figure 10A-B] (10A) Bar graph of IFNγ secretion from isolated CD3 T cells stimulated with anti-CD3 in the presence of constant CD80-Fc levels and soluble CD28 titration. (10B) Isolated PBMCs stimulated with CMV in the presence of constant sCD28 levels and CD80-Fc titration. [Figure 11A-B](11A-B) Line graphs of tumor volume of inoculated H22 cells in immunocompetent mice treated with anti-PD-1 antibody without (11A) and with (11B) administration of recombinant murine CD28. [Figure 12A-C] (12A) Line graph showing antigen binding by serial dilutions of clone M9 to BSA-conjugated CD28 stalk region dimer peptide (right) and recombinant human CD28 protein (left). Antigen was immobilized on a maxisorp ELISA plate. Serial dilutions of clone M9 were performed, and bound antibody was detected using donkey anti-mouse IgG (H&L)-HRP and TMB development. (12B) Bar graph showing ELISA detection of recombinant human sCD28 (left) and sCD28 shed from SEB-activated human PBMCs (right). The ELISA used antibody #3 (2 μg / mL, gray bars) as a positive control, irrelevant antibody M39 (10 μg / mL, dark gray bars) as a negative control, and anti-cleaved antibody M9 (10 μg / mL, black bars). Detection of recombinant CD28 or shed CD28 was performed using the ELISA kit detection antibody conjugated to HRP (0.5 μg / mL). (12C) Histogram showing 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 shown in the gray histogram. Detection was performed by secondary incubation with Alexa Fluor 647-conjugated goat anti-mouse. [Figure 13] Binding to human CD28 stalk region sequences by ELISA. Antigen binding analysis of serial dilutions of different VHH clones. Biotin-conjugated CD28 stalk region dimer peptides, which served as antigens, were immobilized on neutravidin-coated ELISA maxi-sorb plates. Serial dilutions of VHH clones were performed, and bound VHHs were detected with an anti-His tag-HRP conjugated antibody and developed with TMB. [Figure 14]Binding of VHH#2A1 to human membrane 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. [Figure 15] Anti-CD28 stalk region VHH clones do not block ligand binding to membrane CD28. HEK293 cells expressing human CD28 were monitored by flow cytometry for CD86-Fc (2 μg / mL) binding using a secondary anti-human Fc antibody conjugated to Alexa Flour 647. Addition of anti-CD28 VHH clones to CD86-Fc (30 μg / mL, black histogram) did not alter the magnitude of CD86 binding, whereas addition of the commercially available antibody clone CD28.2 (10 μg / mL, top left graph, black histogram) significantly reduced binding. [Figure 16] Evaluation of the agonistic effects of anti-CD28 VHH clones. Human isolated CD3+ cells were stimulated for 2 days with plate-bound anti-CD3 (OKT3, 2 μg / mL, light gray bars) in the presence of the anti-CD28 agonist antibody clone 28.2 (2 μg / mL, dark gray bars), which served as a positive control, an anti-CD28 stalk region VHH, or an irrelevant VHH clone (20 μg / mL, black bars). The concentration of human INFγ in the supernatant was quantified by standardized sandwich ELISA (Biolegend). [Figure 17] In vitro blockade of MMP-2-mediated cleavage of the human CD28 stalk region by VHH clones. c-Myc-bound and biotinylated human CD28 stalk region dimer peptides (1 μM) were incubated with 50 ng of rhMMP-2 for 5 h in the presence of an MMP-2 inhibitor (TMI-1, 50 nM), various concentrations (0.4–10 μg / mL) of M9 Fab, or the indicated VHH clones. The mixture was loaded onto a neutravidin-coated ELISA maxi-sorb plate, followed by extensive washing. Intact peptides were detected with an anti-cMyc HRP-conjugated antibody and developed with TMB. [Figure 18] Anti-CD28 stalk 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 effects of an MMP inhibitor (TMI-1, 1 μM, dark gray bars), an irrelevant VHH negative control (top left graph, black bars), or various concentrations (3.3–100 μg / mL) of anti-CD28 stalk region VHH clones (black bars) on soluble CD28 levels are shown. Soluble human CD28 levels in the supernatant were quantified by a standardized sandwich ELISA (R&D system). [Figure 19] Anti-CD28 stalk region VHH clones 2A1 and 4A4 inhibit CD28 shedding in isolated CD4+ T cells activated with PHA and IL-2. 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 gray bars). The effects of various concentrations (0.4–50 μg / mL), the antibody M9 clone (black bars), an MMP inhibitor (TMI-1, 1 μM, dark gray bars), an irrelevant VHH negative control (top left graph, black bars), and different treatments of anti-CD28 stalk region VHH clones or Fab formats on soluble CD28 levels are shown. Soluble human CD28 levels in the supernatants were quantified by a standardized sandwich ELISA (R&D system). [Figure 20]Anti-CD28 stalk region VHH clones 2A1 and 4A4 inhibit CD28 shedding in superantigen-activated PBMCs. Soluble CD28 levels were measured in the culture medium of isolated PBMCs stimulated with 1 ng / mL SEB (light gray bars). The effects of different treatments on soluble CD28 levels are shown: an MMP inhibitor (TMI-1, 1 μM, dark gray bars), various concentrations (0.4–50 μg / mL) of an irrelevant VHH negative control (top left graph, black bars), anti-CD28 stalk region VHH clones, or the Fab format of the M9 clone (black bars). Soluble human CD28 levels in the supernatants were quantified by a standardized sandwich ELISA (R&D system). [Figure 21] Evaluation of the antagonistic effect of anti-CD28 VHH clones. Human isolated CD3+ cells were stimulated for 24 hours with plate-bound anti-CD3 (OKT3, 2 μg / mL, light gray bars) in the presence of recombinant CD80-Fc protein (5 μg / mL, dark gray bars), which serves as a ligand for CD28 costimulation. An irrelevant VHH clone (top left graph) or anti-CD28 stalk 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 by a standardized sandwich ELISA (Biolegend). [Figure 22] In vitro blocking activity of VHH clone 2A1 against MMP-13-mediated cleavage of the human CD28 stalk region. c-Myc and biotinylated human CD28 stalk region dimer peptide (1 μM) were incubated for 5 h with 50 ng of rhMMP-13 (light gray bars) in the presence of an MMPi (TMI-1, 50 nM, dark gray bars), various concentrations (0.62–10 μg / mL) of an unrelated VHH clone (black bars, left graph), or VHH clone 2A1 (black bars, right graph). The mixture was loaded onto a neutravidin-coated ELISA maxi-sorb plate, followed by extensive washing. Intact peptides were detected with an anti-cMyc-HRP-conjugated antibody and developed with TMB. [Figure 23]Anti-CD28 stalk region VHH clones 2A1, 4A1, and 4A4 specifically bind to the MMP cleavage site of human CD28. Comparison of specific binding of VHH clones to either the human CD28 stalk region WT sequence or the L145K mutant sequence by direct ELISA. Biotin-conjugated wild-type or L145K CD28 stalk region dimer peptides were immobilized on neutravidin-coated ELISA maxi-sorb plates. A dilution series of VHH clones (0.2–5 μg / mL) and an unrelated VHH clone (top left graph) was performed. Bound VHH was detected using an anti-His tag-HRP-conjugated antibody and developed with TMB. DETAILED DESCRIPTION OF THE INVENTION

[0051] In some embodiments, the present invention provides agents of less than 100 kilodaltons (kDa) that bind to membrane CD28 (mCD28) on the cell surface and inhibit the proteolytic cleavage of mCD28. Also provided are methods 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 surprising discovery that full-length antibodies directed against the cleavage site of mCD28 are too large to access the membrane-proximal region and therefore cannot inhibit shedding. Instead, smaller agents with specificity for mCD28 on the cell surface are needed. Furthermore, many cancer patients have elevated levels of sCD28 in their bloodstream caused by sCD28 shedding. This sCD28 functions as an immunosuppressant, so reducing shedding has the dual benefit of reducing sCD28 inhibition and increasing immune activation via mCD28 signaling. Furthermore, it was unexpectedly discovered that sCD28 can inhibit PD-1 / PD-L1-based immunotherapy.

[0052] Drugs According to a first aspect, there is provided an agent that binds to membrane CD28 (mCD28) and inhibits proteolytic cleavage of mCD28.

[0053] In some embodiments, mCD28 is on the cell surface. In some embodiments, mCD28 is within the membrane. In some embodiments, the agent is not a full-length 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 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.

[0054] In some embodiments, the CD28 is mammalian CD28. In some embodiments, the CD28 is human CD28. In some embodiments, the human CD28 comprises or consists of the amino acid sequence: MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 1). In some embodiments, the mature CD28 lacks the signal peptide and comprises the sequence: NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 2).

[0055] In some embodiments, the DNA coding sequence encoding full-length human CD28 comprises the sequence: (SEQ ID NO:3).

[0056] As used herein, sCD28 refers to any CD28 fragment or variant that does not contain a transmembrane domain and therefore cannot be incorporated into a membrane. In some embodiments, the CD28 transmembrane domain comprises 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 the 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 resulting from alternative splicing of exon 3 of CD28. In some embodiments, sCD28 is a cleavage 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 dimeric sCD28. In some embodiments, sCD28 is monomeric sCD28. In some embodiments, sCD28 is not a splice variant resulting from alternative splicing of CD28. In some embodiments, the 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 the 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 the 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.

[0057] In some embodiments, the DNA coding sequence encoding human sCD28 comprises the sequence: (SEQ ID NO:7).

[0058] The effects of sCD28 on immune cells are well known in the art, and non-limiting examples include 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 proinflammatory cytokines such as IL-2 or IFN-γ. In some embodiments, an agent that inhibits proteolytic cleavage of membrane CD28 comprises inhibiting the production of sCD28. In some embodiments, inhibiting the production of sCD28 comprises the inhibitory effect of sCD28 on immune cells.

[0059] As used herein, inhibition of proteolytic cleavage refers to any reduction in proteolytic cleavage of mCD28. In some embodiments, inhibition is at least a 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 99, or 100% reduction in cleavage. Each possibility represents a separate embodiment of the invention. In some embodiments, inhibition of proteolytic cleavage maintains the level of mCD28 on immune cells. In some embodiments, inhibition of proteolytic cleavage increases the level of mCD28 on immune cells. In some embodiments, inhibition of proteolytic cleavage maintains the level of mCD28 appropriate for immune stimulation.

[0060] In some embodiments, the reduction in proteolytic cleavage is a reduction in cleavage by at least one protease. In some embodiments, the reduction in proteolytic cleavage is a reduction in cleavage by at least one metalloprotease. In some embodiments, the metalloprotease is 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, MMP-13, or a combination thereof.

[0061] In some embodiments, the agent is selected from an antigen-binding fragment of an antibody that specifically binds to CD28, a Fab fragment, a single-chain antibody, a single-domain antibody, a small molecule, and a peptide. 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.

[0062] 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 camelid, shark, or nanobody. In some embodiments, the antibody or fragment is fused to another protein or protein fragment. In some embodiments, the second protein or fragment increases half-life, particularly in serum. In some embodiments, the half-life extending protein is human serum albumin. In some embodiments, the agent is modified with a chemical that produces a modification that improves half-life. In some embodiments, the modification is pegylation, and the chemical is polyethylene glycol. It will be understood by those skilled in the art that any half-life extending protein or chemical agent or modification known in the art can be used.

[0063] 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, the agent is 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, the agent is 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 used interchangeably. In some embodiments, the peptide has specific binding to CD28. In some embodiments, the agent is a peptide that has specific binding 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 mimetic" refers to an organic compound capable of specifically binding to a target antigen. In some embodiments, the antibody mimetic is not structurally related to an antibody. Examples of antibody mimics include, but are not limited to, affilins, affimers, affitins, alphabodies, anticalins, avimers, DARPins, finomers, Kunitz domain peptides, monobodies, and nanoCLAMPS. In some embodiments, the antibody mimetic is a DARPin. All of these agents are well known in the art and are known to be useful for blocking interactions between receptors and their ligands. Small molecules and proteins capable of binding to mCD28 can mask the cleavage site or impair or impair access of proteases. In some embodiments, the protein is an antibody mimetic. As used herein, the term "DARPin" refers to a designed ankyrin repeat protein.DARPins are genetically engineered antibody-mimicking proteins that are generally highly specific for protein targets, thus a DARPin for CD28 is one example of a drug.

[0064] In some embodiments, the Fab fragment comprises a size of approximately 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 approximately 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 approximately 15 kDa. In some embodiments, the single-domain antibody comprises a size of 10-17 kDa. In some embodiments, single domain antibodies comprise a size of 10-16 kDa. In some embodiments, single domain antibodies comprise a size of 10-15 kDa. In some embodiments, single domain antibodies comprise a size of 12-15 kDa. In some embodiments, single domain antibodies comprise a size of 12-16 kDa. In some embodiments, single domain antibodies comprise a size of 12-17 kDa. In some embodiments, single domain antibodies comprise a size of less than 25 kDa. In some embodiments, single domain antibodies comprise a size of less than 20 kDa. In some embodiments, single domain antibodies comprise a size of less than 15 kDa. In some embodiments, single domain antibodies comprise a size of 15 kDa or less. Due to their small size and only three CDRs, single domain antibodies comprise a convex shape and bind their epitope from only one side. In comparison, Fab fragments and single-chain antibodies comprise six CDRs and bind epitopes from at least two sides.In some embodiments, binding with only three CDRs allows for superior access to the mCD28 stalk region compared to binding with six CDRs, hi some embodiments, the geometry of single domain antibody binding is superior in terms of access to the mCD28 stalk region.

[0065] As used herein, the term "antibody" refers to a polypeptide or group of polypeptides containing at least one binding domain formed by folding of polypeptide chains, which have an internal surface shape and a three-dimensional binding space with a charge distribution complementary to the characteristics of an antigenic determinant of an antigen. Antibodies typically have a tetrameric form, containing two identical pairs of polypeptide chains, each pair having one "light" and one "heavy" chain. The variable regions of each light / heavy chain pair form the antibody binding site. Antibodies may be oligoclonal, polyclonal, monoclonal, chimeric, camelized, CDR-grafted, multispecific, bispecific, catalytic, humanized, fully human, anti-idiotypic, and soluble or conjugated labeled antibodies, alone or in combination with other amino acid sequences, as well as fragments including epitope-binding fragments, variants, or derivatives thereof. Antibodies may 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 (diabodies), and disulfide-linked variable regions (dsFv). In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site. Antibody fragments may or may not be fused to another immunoglobulin domain, including, but not limited to, an Fc region or fragment thereof. Those skilled in the art will further understand that other fusion products can be generated, including, but not limited to, scFv-Fc fusions, variable region (e.g., VL and VH)-Fc fusions, and scFv-scFv-Fc fusions.

[0066] The immunoglobulin molecule may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass.

[0067] The basic unit of naturally occurring antibody structure is a heterotetrameric glycoprotein complex of approximately 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains, held together by both noncovalent and disulfide bonds. Each heavy and light chain also contains regularly spaced intrachain disulfide bridges. Five human antibody classes exist (IgG, IgA, IgM, IgD, and IgE), and various subclasses within these classes are recognized based on structural differences, such as the number of immunoglobulin units within a single antibody molecule, the disulfide bridge structure of individual units, and 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 of 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 of less than 50 kDa.

[0068] The amino-terminal regions of the heavy and light chains are more diverse in sequence than the carboxy-terminal regions and are therefore called variable domains. This portion of the antibody structure confers antigen-binding specificity to the antibody. The heavy variable (VH) domain and the light variable (VL) domain together form a single antigen-binding site, and therefore a basic immunoglobulin unit has two antigen-binding sites. Specific amino acid residues are thought to form an interface between the light-chain variable domain and the heavy-chain variable domain (Chothia et al., J. Mol. Biol. 186, 651-63 (1985); Novotny and Haber, (1985) Proc. Natl. Acad. Sci. USA 82 4592-4596).

[0069] The carboxy-terminal portions of the heavy and light chains form the constant domains CH1, CH2, CH3, and CL. These domains are much less diverse, but there are differences between animal species, and even within the same individual there are several different antibody isotypes, each with different functions.

[0070] The term "framework region" or "FR" refers to amino acid residues in the variable domain of an antibody other than the hypervariable region amino acid residues as defined herein. The term "hypervariable region", as used herein, refers to amino acid residues in the variable domain of an antibody that are involved in antigen binding. Hypervariable regions contain amino acid residues from "complementarity determining regions" or "CDRs". CDRs are primarily involved in binding to an epitope of an antigen. The extent of FRs and CDRs has been precisely defined (see Kabat et al.).

[0071] Immunoglobulin variable domains can also be analyzed using the IMGT information system (www: / / imgt.cines.fr / ) (IMGT® / V-Quest) to identify variable region segments containing the CDRs. See, e.g., Brochet, X. et al, Nucl. Acids Res. J6:W503-508 (2008).

[0072] also defined a numbering system for variable domain sequences that is applicable to any antibody. One of skill in the art can unambiguously assign this system of "Chothia numbering" to any variable domain sequence without reliance on experimental data beyond the sequence itself. As used herein, "Chothia numbering" refers to the numbering system described in Chothia et al., Journal of Molecular Biology, "Canonical Structures for the Hypervariable Regions of Immunoglobulins" (1987) and Chothia et al., Nature, "Conformations of Immunoglobulin Hypervariable Regions" (1989).

[0073] As used herein, the terms "single-chain antibody" and "single-chain variable fragment" are used interchangeably and refer to a fusion protein of the variable regions of an immunoglobulin heavy and light chains linked by a short peptide linker. In some embodiments, the single-chain antibody has a size of 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 of less than 25 kDa. In some embodiments, the linker of the single-chain antibody is 10-25 amino acids. In some embodiments, the linker is 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 comprises the heavy chain of antibody M9. In some embodiments, the single-chain antibody comprises the light chain of antibody M9. In some embodiments, the single chain antibody comprises the CDRs of antibody M9.

[0074] As used herein, the terms "single domain antibody," "nanobody," and "VHH" are used interchangeably 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. 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.

[0075] Also, as already indicated herein, the amino acid residues of Nanobodies are numbered according to the general numbering scheme for VHs given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, Md., Publication No. 91), which applies to VHH domains from camelids in the article of Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun. 23;240(1-2):185-195. According to this numbering, FR1 of a Nanobody comprises amino acid residues at positions 1 to 30, CDR1 of a Nanobody comprises amino acid residues at positions 31 to 35, FR2 comprises amino acids at positions 36 to 49, CDR2 of a Nanobody comprises amino acid residues at positions 50 to 65, FR3 of a Nanobody comprises amino acid residues at positions 66 to 94, CDR3 of a Nanobody comprises amino acid residues at positions 95 to 102, and FR4 of a Nanobody comprises amino acid residues at positions 103 to 113. In this regard, it should be noted that, as is well known in the art for VH and VHH domains, the total number of amino acid residues in each of the CDRs may vary 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 allowed by the Kabat numbering). This means that in general the Kabat numbering may or may not correspond to the actual numbering of the amino acid residues in the actual sequence.However, in general, it can be said that Kabat numbering indicates that 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, regardless of the number of amino acid residues in the CDR.

[0076] An alternative method for numbering the amino acid residues of VH domains, which can be applied in a similar manner to VHH domains and nanobodies from camelids, is that 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 description, embodiments and figures, unless otherwise stated, the numbering according to Kabat applies to VHH domains according to Riechmann and Muyldermans.

[0077] As used herein, the term "humanized antibody" refers to an antibody from a non-human species in which the protein sequence has been modified to increase similarity to human antibodies. Humanized antibodies can be produced by generating recombinant DNA encoding the CDRs of a non-human antibody surrounded by sequences similar to those of a human antibody. In some embodiments, a humanized antibody is a chimeric antibody. In some embodiments, humanization involves inserting the CDRs of the invention into a human antibody scaffold or framework. Humanized antibodies are well known in the art, and any method for producing them that retains the CDRs of the invention can be used.

[0078] The term "monoclonal antibody" or "mAb," as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies; i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variations that may arise during the generation of the monoclonal antibody, and such variations are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as representing production by any particular method of preparation. The monoclonal antibodies used in accordance with the methods provided herein may be produced by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0079] The mAbs of the present invention may be of any immunoglobulin class, including IgG, IgM, IgD, IgE, or IgA. Hybridomas producing mAbs can be cultured in vitro or in vivo. High-titer mAbs can be obtained by in vivo production, and cells from individual hybridomas are injected intraperitoneally into pristine-primed Balb / c mice to produce ascites fluid containing high concentrations of the desired mAb. mAbs of the IgM or IgG isotype can be purified from such ascites fluid or from the culture supernatant using column chromatography methods well known to those skilled in the art.

[0080] "Antibody fragments" include portions of intact antibodies, preferably including the antigen-binding region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, tandem diabodies (taDbs), linear antibodies (e.g., U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062(1995)), one-arm antibodies, single variable domain antibodies, minibodies, single-chain antibody molecules, multispecific antibodies formed from antibody fragments (e.g., Db-Fc, taDb-Fc, taDb-CH3, (scFV)4-Fc, di-scFv, bi-scFv, or tandem (di, tri)-scFv), and bispecific T-cell engagers (BiTEs).

[0081] 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, the name reflecting its ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0082] An "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy- and one light-chain variable domain tightly covalently bound. Three surfaces of the VH-VL dimer are in this configuration. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although with lower affinity than the entire binding site.

[0083] Fab fragments also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' in which the cysteine residues of the constant domains bear 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.

[0084] The "light chains" of antibodies (immunoglobulins) from vertebrate species can be assigned to one of two clearly distinguishable types, called kappa and lambda, based on the amino acid sequences of their constant domains.

[0085] Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to the different classes of antibodies are called a, δ, e, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0086] "Single-chain Fv" or "scFv" antibody fragments comprise 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 comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see Pluckthun, "The Pharmacology of Monoclonal Antibodies," vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0087] The term "diabody" refers to a small antibody fragment having two antigen-binding sites, comprising a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) of the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains of the same chain, the domains pair with complementary domains of another chain to form two antigen-binding sites. The production of diabodies is known in the art and is described in Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0088] 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 in which the VHVL unit comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) with polyepitopic specificity, antibodies with two or more VL and VH domains in which each VHVL unit binds a different epitope, antibodies with two or more single variable domains in which each single variable domain binds a different epitope, full-length antibodies, Fabs, Fvs, dsFvs, scFvs, diabodies, bispecific diabodies, triabodies, trifunctional antibodies, and antibody fragments such as 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.

[0089] The monoclonal antibodies of the present invention can be prepared using methods well known in the art, including various techniques such as those described in Kohler, G. and Milstein, C., Nature 256:495-497 (1975), Kozbor et al., Immunology Today 4:72 (1983), and Cole et al., pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

[0090] In addition to traditional methods for producing antibodies in vivo, antibodies can also be generated in vitro using phage display technology. The generation of such recombinant antibodies is much faster than traditional antibody generation, and they can be generated against a vast number of antigens. Furthermore, using traditional methods, many antigens are known to be non-immunogenic or highly toxic, making them unsuitable for antibody production in animals. Furthermore, affinity maturation of recombinant antibodies (i.e., increasing affinity and specificity) is very simple and relatively fast. Finally, a large number of different antibodies against a particular antigen can be generated in a single selection procedure. To generate recombinant monoclonal antibodies, various display library-based methods can be used to generate large pools of antibodies with different antigen recognition sites. Such libraries can be created in several ways: by generating synthetic repertoires by cloning synthetic CDR3 regions into a pool of heavy chain germline genes, a large antibody repertoire can be generated, from which recombinant antibody fragments with various specificities can be selected. Human lymphocyte pools can be used as starting material for antibody library construction. It is possible to construct naive repertoires of human IgM antibodies, thus creating highly diverse human libraries. This method has been widely and successfully used to select large numbers of antibodies against a variety of antigens. Protocols for the construction of bacteriophage libraries and the selection of recombinant antibodies are described 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.

[0091] Non-human antibodies can be humanized by any method known in the art. In one method, non-human complementarity determining regions (CDRs) are inserted into a human antibody or consensus antibody framework sequence. Further modifications can then be introduced into the antibody framework to adjust affinity or immunogenicity.

[0092] In some embodiments, antibodies and portions 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 the group consisting of an Fv, Fab, F(ab')2, scFv, or scFv2 fragment.

[0093] In some embodiments, the invention provides nucleic acid sequences encoding the antibodies or antigen-binding portions of the invention.

[0094] For example, a polynucleotide can encode an entire 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). Typically, it includes three constant regions, CH1, CH2, and CH3, as well as a "hinge" region. In some situations, the presence of a constant region is desirable.

[0095] Other polypeptides that can be encoded by the polynucleotides include antigen-binding antibody fragments such as single-domain antibodies ("dAbs"), Fvs, scFvs, Fab's, and CHIs, in which the CK or CL domains have been truncated. Because minibodies are smaller than conventional antibodies, they should have improved tissue penetration for clinical / diagnostic uses, but because they are bivalent, they should retain higher binding affinity than monovalent antibody fragments such as dAbs. Thus, unless otherwise indicated by context, the term "antibody," as used herein, encompasses not only whole antibody molecules but also antigen-binding antibody fragments of the types discussed above. Each framework region present in the encoded polypeptide can contain at least one amino acid substitution compared to the corresponding human acceptor framework. Thus, for example, a framework region can contain a total of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions relative to the acceptor framework region. Given the properties of the individual amino acids that make up the disclosed protein products, some reasonable substitutions will be recognized by those skilled in the art. 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.

[0096] Suitably, the polynucleotides described herein may be isolated and / or purified. In some embodiments, the polynucleotides are isolated polynucleotides.

[0097] As used herein, "non-naturally occurring" substances, compositions, entities, and / or any combination of substances, compositions or entities, or grammatical variations thereof, is a conditional term that expressly excludes, but only excludes, those forms of substances, compositions, entities, and / or any combination of substances, compositions or entities that are well understood by those of ordinary skill in the art as "naturally occurring" or that are or can be determined or interpreted as "naturally occurring" by a determining person or governmental or judicial body at any time.

[0098] Another aspect provides an agent comprising three CDRs, wherein 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).

[0099] Another aspect provides an agent comprising three CDRs, wherein 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).

[0100] Another aspect provides an agent comprising three CDRs, wherein 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: 39 (AITSGGSTNYADSVKG), and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 40 (DLYGEDYWI).

[0101] In some embodiments, the CDRs are numbered according to the Abm numbering system. In some embodiments, the CDRs are numbered according to the Chothia numbering system. In some embodiments, the CDRs are numbered according to the Kabat numbering system.

[0102] In some embodiments, CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 41 (INAMX1), where X1 is G or A. In some embodiments, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 42 (AIX1X2X3GX4TX5YAX6SVKG), where 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), where 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), where X1 is E or L and X2 is D or I.

[0103] 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 comprise any other CDRs other than those listed above.

[0104] In some embodiments, the agent comprises a sequence comprising and / or consisting of EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVTVSS (SEQ ID NO: 30).

[0105] In some embodiments, the agent comprises a sequence comprising and / or consisting of EVQLVESGGGLVQAGGSLRLSCAASGSLFSINAMAWYRQAPGKQRELVAAITSSGSTNYANSVKGRFTVSRDNAKNTMYLQMNSLKPEDTAVYYCVVDEYGSDYWIWGQGTQVTVSS (SEQ ID NO: 31).

[0106] In some embodiments, the agent comprises a sequence comprising and / or consisting of QVQLVESGGGLVQAGGSLRLSCAASGSIFSINAMGWYRQAPGKQRERVAAITSGGSTNYADSVKGRFTISRDNAKNTVYLQMNNLEPRDAGVYYCVVDLYGEDYWIWGQGTQVTVSS (SEQ ID NO: 32).

[0107] In some embodiments, the VHH sequence further comprises 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 linked to the VHH via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is an alanine repeat linker. In some embodiments, the alanine repeat comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues. Each possibility represents a separate embodiment of the present invention. In some embodiments, the alanine repeat linker consists of 3 alanine residues. In some embodiments, the His tag is a 6His tag.

[0108] In some embodiments, VHH sequences that have been found to specifically bind to the stalk region of human CD28 and contain a His tag are EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVTVSSAAAHHHHHH (SEQ ID NO: 45, clone 2A1), EVQLVESGGGLVQAGGSLRLSCAASGSLFSINAMAWYRQAPGKQR ELVAAITSSGSTNYANSVKGRFTVSRDNAKNTMYLQMNSLKPEDTAVYYCVVDEYGSDYWIWGQGTQVTVSSAAAHHHHHH (SEQ ID NO: 46, clone 4A4), and QVQLVESGGGLVQAGGSLRLSCAASGSIFSINAMGWYRQAPGKQRERVAAITSGGSTNYADSVKGRFTISRDNAKNTVYLQMNNLEPRDAGVYYCVVDLYGEDYWIWGQGTQVTVSSAAAHHHHHH (SEQ ID NO: 47, clone 4A1).

[0109] Another embodiment provides an agent comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 11 (GYTLTNY), CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 12 (NTYTGK), CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 13 (GDANQQFAY), CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 14 (KASQDINSYLS), CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 15 (RANRLVD), and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 16 (LQYDEFPPT). This antibody is referred to herein as M9.

[0110] In some embodiments, the agent comprises three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 17 (GFTFSSYYMS), CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 18 (TISDGGDNTYYAGTVTG), CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 19 (IHWPYYFDS), CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 20 (RASSSVSYMN), CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 21 (ATSDLAS), and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 22 (QQWSSHPPT).

[0111] In some embodiments, the agent comprises a heavy chain comprising the 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 comprises a heavy chain comprising a polypeptide encoded by the nucleic acid sequence GACGTGAAGCTCGTGGAGTCTGGGGGAGGCTTAGTGAAGCTTGGAGGGTCCCTGAAACTCTCCTGTGTAGCCTCTGGATTCACTTTCAGTAGCTATTACATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCGACCATAAGTGATGGTGGTGATAACACCTACTACGCAGGCACTGTGACGGGCCGATTCACCATCTCCAGAGACTTTGCCAAGAACACCCTGTACCTGCAAATGAACAGTCTGACCTCTGAGGACACAGCCGTGTATTACTGTGCAAGAATTCATTGGCCTTACTATTTTGACTCCTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO:24). In some embodiments, the heavy chain consists of SEQ ID NO:24. Antibody M9 has been sequenced and found to have a heavy chain consisting of SEQ ID NO:24. The CDRs of this heavy chain, determined using the Chothia scheme, are SEQ ID NOs: 17-19.

[0112] In some embodiments, the agent comprises a light chain comprising the amino acid sequence QFVLSQSPAILSASPGEMLTMTCRASSSVSYMNWYQQKPGSSPKPWIYATSDLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSHPPTFGGGTKLEIR (SEQ ID NO: 25). In some embodiments, the variable region of the light chain comprises and / or consists of SEQ ID NO: 25. In some embodiments, the agent comprises a light chain comprising a polypeptide encoded by the nucleic acid sequence CAATTTGTTCTCTCCCAGTCTCCAGCAATCCTGTCTGCATCTCCCGGGGAGATGCTCACAATGACTTGCAGGGCCAGCTCAAGTGTAAGTTATATGAACTGGTATCAGCAGAAGCCAGGATCTTCCCCCAAACCCTGGATTTATGCCACATCCGACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTATTCTCTCACAATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTCACCCACCCACGTTCGGAGGGGGGACCAAGCTGGAAATAAGA (SEQ ID NO: 26). In some embodiments, the light chain consists of SEQ ID NO: 26. Antibody M9 has been sequenced and found to have a light chain consisting of SEQ ID NO: 26. The CDRs of this light chain, determined using the Chothia scheme, are SEQ ID NOs: 20-22.

[0113] 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.

[0114] 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.

[0115] The term "agonist" generally refers to a molecule, compound, or agent that binds to a receptor and fully or partially activates the receptor. In some embodiments, an agonist binds at the same site as the natural ligand. In some embodiments, an agonist binds at an allosteric site that is different from the binding site of the natural ligand. The term "antagonist" generally refers to a molecule, compound, or agent that binds to a receptor at the same site as an agonist or at a different site, does not activate the receptor, and prevents or blocks activation of the receptor by the natural ligand and prevents or blocks activation of the receptor by a receptor agonist. In some embodiments, antibodies of the invention bind to mCD28 but do not activate or block receptor activation. In some embodiments, they do not block activation by CD86. In some embodiments, antibodies of the invention do not bind to mCD28.

[0116] As used herein, a "direct agonist / antagonist" refers to a molecule that binds to a receptor (mCD28) and thereby increases / decreases signaling by that molecule. In the case of mCD28, an agonist increases mCD28 signaling in cells by binding to mCD28. 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 decreases mCD28 signaling in cells by binding to mCD28. 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 a ligand, an inhibitor, a co-receptor, or a molecule other than the receptor in question to alter receptor signaling are not considered direct agonists / antagonists. In some embodiments, the agents of the present invention contact sCD28 in serum, thereby allowing increased signaling through mCD28 on cells. As a result, mCD28 signaling is increased, but the antibody is not an mCD28 agonist or direct agonist because binding to mCD28 does not increase receptor signaling.

[0117] In some embodiments, the agent does not bind to the ligand-binding domain of mCD28. In some embodiments, the agent does not mask or block access to the ligand-binding domain. In some embodiments, the agent does not bind to, mask or block access to the IgV domain of sCD28. In some embodiments, the IgV domain is the 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 amino acid sequence MLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKG (SEQ ID NO: 8). In some embodiments, the agent does not inhibit binding of sCD28 to a 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, the CD86 is CD86-Fc. In some embodiments, the CD80 is CD80-Fc.

[0118] In some embodiments, the agent binds to the stalk region of CD28. In some embodiments, the agent binds to the membrane proximal region of mCD28. In some embodiments, the stalk region comprises the sequence GKHLCPSPLFPGPSKP (SEQ ID NO: 9). In some embodiments, the stalk region comprises the sequence KGKHLCPSPLFPGPS (SEQ ID NO: 27). In some embodiments, the stalk 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 but not dimeric CD28. In some embodiments, the fragment of the CD28 extracellular domain is the stalk region. In some embodiments, an agent that binds to CD28 prevents cleavage of CD28. In some embodiments, the agent that binds to CD28 prevents CD28 from being shedding from cells.

[0119] In some embodiments, the agent binds to a cleavage site in the stalk region. In some embodiments, the agent binds to a cleavage site in mCD28. In some embodiments, the agent binds to a cleavage site of at least one protease. In some embodiments, the agent binds to a cleavage site of MMP-2.

[0120] In some embodiments, the agent does not bind to the ligand binding domain of mCD28. In some embodiments, the agent does not shield or block access to the ligand binding domain. In some embodiments, the agent binds to the cleavage site. In some embodiments, the agent masks, shields, or blocks access to the cleavage site. In some embodiments, the agent binds, blocks, shields, or masks the protease cleavage site. In some embodiments, the agent does not bind to the protease cleavage site but masks it. In some embodiments, the agent blocks access to the protease cleavage site. In some embodiments, the agent creates a steric hindrance that blocks the protease cleavage site. In some embodiments, the agent does not bind to the protease cleavage site, but binding of the agent creates a conformational change in mCD28 that blocks the protease cleavage site. In some embodiments, binding of the agent creates 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 PSPL in the stalk domain of mCD28.

[0121] 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.

[0122] In some embodiments, the agent does not bind to the stalk region of CD28 having a mutated cleavage site. In some embodiments, the stalk region of CD28 having a mutated cleavage site is not a substrate for proteases. In some embodiments, the stalk region of CD28 having a mutated cleavage site is not a substrate for metalloproteases. In some embodiments, the stalk region of CD28 having a mutated cleavage site is not a substrate for matrix metalloproteases. In some embodiments, the stalk region of CD28 having a mutated cleavage site is not a substrate for matrix metalloprotease 2 (MMP-2). In some embodiments, the stalk region of CD28 having a mutated cleavage site is not a substrate for matrix metalloprotease 13 (MMP-13). In some embodiments, the mutated cleavage site is a mutation of leucine 145 in SEQ ID NO: 1. In some embodiments, the mutated cleavage site is an amino acid substitution of leucine 145 in SEQ ID NO: 1. In some embodiments, the amino acid substitution of leucine 145 in SEQ ID NO: 1 is lysine.

[0123] In some embodiments, the agent does not modulate CD28 function and / or signaling. In some embodiments, the agent does not degrade mCD28. In some embodiments, the agent does not lead to or promote mCD28 degradation. In some embodiments, the signaling 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 internalization or recycling of the CD28 receptor. Costimulation via mCD28 is essential for T cell immune activation. Proteolytic cleavage removes the ligand-binding domain in the extracellular region of CD28 from the transmembrane and cytoplasmic portions of the protein that remain in the membrane. Thus, cleaved CD28 cannot signal and cannot contribute to T cell activation. Therefore, agents that block cleavage and are both antagonists do not allow mCD28 activation. Similarly, agents that block cleavage but are also agonists may induce aberrant T cell activation and potentially autoimmune responses.

[0124] In some embodiments, the agent does not reduce surface levels of mCD28 on immune cells. In some embodiments, the immune cells are T cells. In some embodiments, the agent reduces surface levels 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.

[0125] In some embodiments, binding of an agent to a cell does not kill the cell. In some embodiments, binding of an agent to a cell does not lead to cell death. In some embodiments, the agent does not induce antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the agent does not induce complement-dependent cytotoxicity (CDC). In some embodiments, the agent does not induce ADCC and / or CDC. In some embodiments, the agent is an antibody and comprises an IgG2 or IgG4 domain. In some embodiments, the antibody comprises an IgG2 domain. In some embodiments, the antibody comprises an IgG4 domain. In some embodiments, the antibody comprises an IgG1 or IgG3 mutated to reduce cell death mediated by antibody binding. 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.

[0126] In some embodiments, the agent lacks an Fc domain. In some embodiments, the agent is an antigen-binding domain that lacks an Fc domain. In some embodiments, the agent is a single-domain antibody. In some embodiments, the agent is a camelid, shark, or nanobody.

[0127] 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 antibody mimetic has a molar mass of less than 100, 90, 80, 70, 60, 50, 40, 30, or 20 kDa. Each possibility represents a separate embodiment of the 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 mimetics include, but are not limited to, affilins, affimers, affitins, alphabodies, anticalins, avimers, DARPins, finomers, Kunitz domain peptides, monobodies, and nanoCLAMPS. In some embodiments, the antibody mimetic is a DARPin.

[0128] In some embodiments, the agent inhibits proteolytic 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 acid protease. In some embodiments, the protease is a glutamic acid protease. In some embodiments, the protease is selected from aspartic acid, glutamic acid, serine, cysteine, and threonine proteases. In some embodiments, the protease is an asparagine peptide lyase. In some embodiments, the protease is a 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 metalloprotease is matrix metalloprotease MMP-13 (MMP-13). In some embodiments, the metalloprotease is ADAM10. In some embodiments, the metalloprotease is ADAM17. In some embodiments, the metalloprotease is ADAM10, MMP-2, and / or ADAM17. In some embodiments, the metalloprotease is ADAM10, MMP-2, MMP-13, and / or ADAM17.In some embodiments, the metalloprotease is MMP-2, ADAM10, ADAM17, or a combination thereof. In some embodiments, the metalloprotease is MMP-2, MMP-13, ADAM10, ADAM17, or a combination thereof.

[0129] How to use 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 invention.

[0130] According to another aspect, there is provided a method of improving immunotherapy in a subject in need thereof, the method comprising administering an agent of the invention.

[0131] According to another aspect, there is provided a method of reducing sCD28 in a subject in need thereof, the method comprising administering an agent of the invention.

[0132] In some embodiments, the immunotherapy is PD-1 and / or PD-L1 based immunotherapy. In some embodiments, the PD-1 / PD-L1 based immunotherapy comprises administering an anti-PD1 or anti-PD-L1 antibody. In some embodiments, the therapy comprises blockade of the PD-1 checkpoint. In some embodiments, the immunotherapy comprises administering allogeneic, syngeneic, 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 is suffering from cancer. In some embodiments, the subject is suffering from cancer. In some embodiments, the cancer is an sCD28-positive cancer. In some embodiments, the cancer is a high sCD28 cancer. In some embodiments, the subject is at risk of developing cancer.

[0133] As used herein, the term "treatment" or "treating" of a disease, disorder, or condition includes alleviating at least one symptom thereof, reducing its severity, or inhibiting its progression. Treatment does not necessarily mean that the disease, disorder, or condition is completely cured. To be an effective treatment, a composition useful herein need only reduce the severity of the disease, disorder, or condition, reduce the severity of symptoms associated therewith, or provide an improvement in the quality of life of the patient or subject.

[0134] In some embodiments, reducing comprises administering at least one agent of the present invention to the subject. As used herein, the terms "administering," "administration," and the like refer to any method, in sound medical practice, of delivering a composition containing an active agent to a subject in a manner that provides a therapeutic effect. One aspect of the present subject matter provides for oral administration of a therapeutically effective amount of an agent of the present invention to a patient in need thereof. Other suitable routes of administration may include parenteral, subcutaneous, intravenous, intramuscular, or intraperitoneal.

[0135] According to another aspect, there is provided a pharmaceutical composition comprising an agent of the invention and a therapeutically acceptable carrier, adjuvant, or excipient. In some embodiments, administering is administering a pharmaceutical composition of the invention.

[0136] As used herein, the terms "carrier," "excipient," or "adjuvant" refer to any ingredient 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, formulation auxiliary of any type, or simply a sterile aqueous medium such as physiological 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; powdered tragacanth; 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, and soybean oil; glycols such as propylene glycol, polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol and phosphate buffers, and other non-toxic 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 oil, polyol, alginic acid, pyrogen-free water, isotonic saline, phosphate buffer, cocoa butter (suppository base), emulsifier, and other non-toxic pharmaceutically acceptable materials used in other pharmaceutical preparations.Wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, excipients, stabilizers, antioxidants, and preservatives can also be present.Non-toxic, inert, and effective carriers can be used to formulate the compositions contemplated herein.In this regard, suitable pharmaceutically acceptable carriers, excipients and diluents are well known to those skilled in the art, and can be found in, for example, 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, all of whose contents are incorporated herein by reference in their entirety.Examples of pharmaceutically acceptable carriers, carriers and diluents useful in the present composition include distilled water, physiological saline, Ringer's solution, dextrose solution, Hank's solution and DMSO. These additional inactive ingredients, as well as effective formulation and administration procedures, are well known in the art and are 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 contained in artificially engineered structures such as liposomes, ISCOMS, sustained-release particles, and other vehicles that increase the half-life of peptides or polypeptides in serum. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. Liposomes for use with the peptides described herein are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The choice of lipid is generally determined by considerations such as liposome size and stability in the blood. Various methods for preparing liposomes are available, as discussed, for example, by Coligan, JE et al., Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York; see also U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0137] Carriers in total may comprise from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0138] In some embodiments, the methods of the present invention do not degrade or lead to the 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 the subject's immune cells. In some embodiments, the methods of the present invention increase mCD28 levels on the subject's immune cells.

[0139] In some embodiments, the reduction is at least a 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% reduction in sCD28. Each possibility represents a separate embodiment of the present invention. In some embodiments, the reduction is a reduction in serum sCD28. In some embodiments, the reduction is a reduction in blood levels of sCD28. In some embodiments, the reduction is a reduction in sCD28 levels in the tumor microenvironment (TME).

[0140] In some embodiments, the subject's blood contains elevated levels of sCD28. In some embodiments, the subject's blood before reduction contains elevated levels of sCD28. In some embodiments, the levels are elevated above levels in healthy subjects. In some embodiments, the subject's sCD28 levels are elevated by 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 levels in healthy subjects. Each possibility represents a separate embodiment of the present invention. In some embodiments, levels are elevated to greater than 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, levels are elevated to greater than 5 ng / ml. In some embodiments, levels are elevated to greater than 10 ng / ml. In some embodiments, levels are elevated to greater than 20 ng / ml. In some embodiments, the subject's blood comprises at least 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45, or 50 ng of sCD28 per ml of blood. Each possibility represents a separate embodiment of the present invention. In some embodiments, the subject's blood before depletion contains at least 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45, or 50 ng of sCD28 per ml of blood. Each possibility represents a separate embodiment of the present invention. In some embodiments, the subject's blood contains at least 5 ng / ml of sCD28. In some embodiments, the subject's blood contains at least 10 ng / ml of sCD28. In some embodiments, the subject's blood contains at least 20 ng / ml of sCD28. In some embodiments, the subject's blood before depletion contains at least 5 ng / ml of sCD28. In some embodiments, the subject's blood before depletion contains at least 10 ng / ml of sCD28.In some embodiments, the subject's blood before depletion contains at least 20 ng / ml of sCD28.

[0141] In some embodiments, the subject is suffering from cancer. In some embodiments, the cancer is a cancer that can be treated with PD-1 / PD-L1 therapy. In some embodiments, the subject is receiving 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 is naive to PD-1 / PD-L1 therapy. In some embodiments, the methods of the present invention are performed in conjunction with PD-1 / PD-L1 therapy. In some embodiments, the methods of the present invention are performed before PD-1 / PD-L1 therapy.

[0142] In some embodiments, the method further comprises administering to the subject another immunotherapy. In some embodiments, the method further comprises administering a PD-1 and / or PD-L1 based immunotherapy. In some embodiments, the other immunotherapy is a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is a PD-1 and / or PD-L1 inhibitor. In some embodiments, the checkpoint inhibitor is a CTLA-4 inhibitor. In some embodiments, the other immunotherapy is a chimeric antigen receptor (CAR) based immunotherapy. 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.

[0143] As used herein, the terms "CAR-T cells" and "CAR-NK cells" refer to engineered receptors that have specificity for at least one protein of interest (e.g., an immunogenic protein whose expression is increased after treatment with an epigenetic modifier) and are grafted onto immune effector cells (T cells or NK cells). In some embodiments, the CAR-T cells have the specificity of a monoclonal antibody grafted onto a T cell. In some embodiments, the CAR-NK cells have the specificity of a monoclonal antibody grafted onto an NK cell. In some embodiments, the T cells are selected from cytotoxic T lymphocytes and regulatory T cells.

[0144] CAR-T and CAR-NK cells and their vectors are well known in the art. Such cells target proteins bound by receptors and exhibit cytotoxicity. 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 whose expression is increased by contact with an epigenetic modifier.

[0145] The construction of CAR-T cells is well known in the art. In one non-limiting example, a monoclonal antibody against a viral protein can be generated, followed by the construction of a vector encoding the antibody. The vector also includes a costimulatory signal region. In some embodiments, the costimulatory signal region includes the intracellular domain of a known T cell or NK cell stimulatory 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, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83. In some embodiments, the vector also includes a CD3Z signaling domain. This vector is then transfected into T cells, for example, by lentiviral infection.

[0146] In some embodiments, the cancer is a cancer with elevated sCD28 levels. In some embodiments, the cancer comprises 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. Each possibility represents a separate embodiment of the present invention. In some embodiments, the cancer comprises 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, the 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, renal 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, and colorectal cancer. In some embodiments, the cancer is melanoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, renal cancer, gastric cancer, or colorectal cancer. Each possibility represents a separate embodiment of the present invention.

[0147] kit According to another aspect, there is provided a kit comprising at least one agent of the invention, or a pharmaceutical composition of the invention.

[0148] In some embodiments, the kit further comprises a PD-1- and / or PD-L1-based immunotherapeutic. In some embodiments, the kit comprises a label indicating that the agent of the invention is for use in conjunction with a PD-1- and / or PD-L1-based immunotherapeutic. In some embodiments, the kit comprises a label indicating that the PD-1- and / or PD-L1-based therapeutic is for use in conjunction with an antibody or pharmaceutical composition of the invention.

[0149] Another aspect provides a kit containing a PD-1- and / or PD-L1-based immunotherapeutic, the kit including a label indicating that the immunotherapeutic is for use with an antibody or pharmaceutical composition of the invention.

[0150] In some embodiments, the kits of the invention are for use in treating cancer. In some embodiments, the kits of the invention are diagnostic kits. In some embodiments, the kits of the invention are for use in determining the serum level of sCD28 in a subject in need thereof. In some embodiments, the subject is afflicted with cancer. In some embodiments, the kits of the invention are for use in determining the suitability of a subject for treatment with an agent or pharmaceutical composition of the invention. In some embodiments, the kits are for use in determining the suitability of a subject for treatment with anti-PD-1 / PD-L1-based immunotherapy.

[0151] Method of drug production According to another aspect, there is provided a method for producing an agent that inhibits proteolytic cleavage of mCD28 on the cell surface, comprising: a. Obtaining an agent that binds to the CD28 extracellular domain or a fragment thereof, wherein the agent is less than 100 kDa; b. Testing the binding of the obtained agent to mCD28 on the cell surface; and c. selecting an agent that binds to cell surface mCD28; Including, Thereby, an agent is generated that inhibits the proteolytic cleavage of mCD28 on the cell surface.

[0152] According to another aspect, there is provided a method for producing an agent that inhibits proteolytic cleavage of mCD28 on the cell surface, comprising: a. Culturing a host cell containing one or more vectors comprising a nucleic acid sequence encoding an agent, wherein the nucleic acid sequence is i. Obtaining an agent that binds to the CD28 extracellular domain or a fragment thereof, wherein the agent is less than 100 kDa; ii. Testing the binding of the obtained agent to mCD28 on the cell surface; and iii. selecting an agent that binds to cell surface mCD28; and culturing the host cells containing the selected agent's nucleic acid sequence; Including, Thereby, an agent is generated that inhibits the proteolytic cleavage of mCD28 on the cell surface.

[0153] In some embodiments, the method further comprises testing the ability of the agent to block cleavage of mCD28 on the cell surface by a protease. In some embodiments, the agent is an anti-cleavage agent. In some embodiments, the agent is an anti-shedding agent. In some embodiments, the agent reduces shedding of sCD28 in a subject. In some embodiments, the agent reduces cleavage of mCD28. In some embodiments, the agent reduces cleavage of mCD28 in a subject.

[0154] 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.

[0155] As used herein, the term "extracellular domain of CD28" refers to the N-terminal portion of CD28 preceding 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 stalk domain. In some embodiments, the extracellular domain of CD28 comprises the stalk domain of CD28. In some embodiments, the extracellular domain of CD28 comprises or consists of the sequence NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 28). In some embodiments, the extracellular domain of CD28, or a fragment thereof, is dimeric. 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.

[0156] As used herein, a "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, obtaining an agent that binds to a fragment of the extracellular domain of CD28 refers to obtaining an agent that specifically binds to the CD28 stalk domain.

[0157] In some embodiments, the method further comprises assaying mCD28 downstream signaling in the presence of the obtained agent and selecting at least one agent that does not substantially agonize or substantially antagonize mCD28 signaling. In some embodiments, the selecting comprises selecting at least one agent that does not antagonize mCD28 signaling. It will be understood by those skilled in the art that stimulating CD28 signaling for the treatment of cancer may not be harmful, but antagonizing signaling may be counterproductive.

[0158] In some embodiments, testing the ability of an agent to block cleavage comprises measuring sCD28 in the serum of activated immune cells in the presence and absence of the agent. In some embodiments, testing the ability of an agent to block cleavage comprises combining the agent, a protease, and the extracellular domain of CD28 or a fragment thereof comprising the cleavage site. In some embodiments, testing further comprises sequencing the extracellular domain of CD28 or a fragment thereof to check for truncation and / or cleavage. In some embodiments, testing further comprises running the extracellular domain of CD28 or a fragment thereof on a gel sensitive enough to measure size changes due to cleavage. In some embodiments, testing further comprises measuring the production of sCD28 from cells expressing mCD28 in the presence of the agent and a protease.

[0159] In some embodiments, obtaining the agent comprises immunizing a shark or camelid with the CD28 extracellular domain or fragment thereof and collecting antibodies from the immunized organism, hi some embodiments, obtaining the agent comprises screening a library of agents for binding to the CD28 extracellular domain or fragment thereof and selecting agents that bind.

[0160] In some embodiments, collecting the antibodies comprises extracting B cells from the spleen of the immunized shark or camelid. In some embodiments, the B cells are fused with melanoma cells to generate hybridomas. In some embodiments, the antibodies are collected from the culture medium of the hybridoma. In some embodiments, obtaining the agent comprises immunizing an organism with the CD28 extracellular domain or a fragment thereof and collecting the antibodies from the immunized organism. In some embodiments, the organism is a mouse. In some embodiments, the organism is selected from a rabbit, a mouse, a rat, a shark, a camelid, a chicken, a goat, and a phage. In some embodiments, the camelid is selected from a camel and a llama. In some embodiments, collecting comprises drawing blood. In some embodiments, collecting comprises Extracting B cells from the spleen of an immunized organism; b. fusing the extracted B cells with myeloma cells to produce hybridomas; and C. Harvesting antibodies from hybridomas Includes:

[0161] In some embodiments, obtaining the agent comprises screening a library of agents for binding to the CD28 extracellular domain or a fragment thereof and selecting an agent that does so. In some embodiments, the library is a phage display library. In some embodiments, the library is an immunization library derived from splenic 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 library of VHH antibodies. In some embodiments, the library is a library of single chain, single domain, or nanobodies. In some embodiments, obtaining the agent comprises sequencing the agent. In some embodiments, obtaining the agent comprises generating a recombinant form of the agent. In some embodiments, selecting the agent comprises sequencing the agent. In some embodiments, selecting the agent comprises generating a recombinant form of the agent. In some embodiments, the recombinant form is generated from the sequence of the agent. In some embodiments, the method further comprises humanizing the agent.

[0162] It is well known to those skilled in the art to express a nucleic acid molecule encoding a drug in a cell. This can be achieved by transfection, viral infection, or direct modification of the cell's genome, among other methods. In some embodiments, the gene is in an expression vector such as a plasmid or viral vector. One such example of an expression vector containing p16-Ink4a is the mammalian expression vector pCMV p16 INK4A available from Addgene.

[0163] A vector nucleic acid sequence generally includes at least an origin of replication for propagation within a cell, and optionally additional elements such as heterologous polynucleotide sequences, expression control elements (e.g., promoters, enhancers), selectable markers (e.g., antibiotic resistance), polyadenine sequences, etc.

[0164] The vector may be a DNA plasmid delivered by non-viral or viral methods.The viral vector may be a retroviral vector, a herpesvirus vector, an adenovirus vector, an adeno-associated virus vector, or a poxvirus vector.The promoter may be active in mammalian cells.The promoter may be a viral promoter.

[0165] In some embodiments, the nucleic acid sequence encoding the agent is operably linked to a promoter. The term "operably linked" is intended to mean that the nucleotide sequence of interest is linked to regulatory elements in a manner that allows expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell).

[0166] In some embodiments, the vector is introduced into cells 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-velocity ballistic penetration by small particles that contain nucleic acid either within the matrix of the small beads or particles or on their surface (Klein et al., Nature 327, 70-73 (1987)), and / or the like.

[0167] The term "promoter," as used herein, refers to a group of transcriptional control modules clustered around the initiation site of RNA polymerase, i.e., RNA polymerase II. Promoters are composed of individual functional modules, each consisting of approximately 7-20 bp of DNA and containing one or more recognition sites for transcriptional activator or repressor proteins.

[0168] In some embodiments, the nucleic acid sequence is transcribed by RNA polymerase II (RNAP II and Pol II). RNAP II is an enzyme found in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNAs and microRNAs.

[0169] 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 (available from Invitrogen), pCI (available from Promega), pMbac, pPbac, pBK-RSV and pBK-CMV (available from Strategene), pTRES (available from Clontech), and derivatives thereof.

[0170] In some embodiments, expression vectors containing regulatory elements from eukaryotic viruses, such as retroviruses, are used in accordance with the present invention. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papillomavirus 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 vector that allows protein expression under the direction of the SV-40 early promoter, SV-40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown to be effective for expression in eukaryotic cells.

[0171] In some embodiments, recombinant viral vectors that offer advantages such as horizontal infection and target specificity are used for in vivo expression. In one embodiment, horizontal infection, for example, is inherent in the retroviral life cycle, a process in which a single infected cell produces many progeny virions that bud and infect neighboring cells. In one embodiment, this results in the rapid infection of a large area, most of which was not initially infected by the original viral particle. In one embodiment, a viral vector that cannot spread horizontally is generated. In one embodiment, this feature can be useful when the desired goal is to introduce a specific gene into only a localized number of target cells.

[0172] A variety of methods can be used to introduce the expression vectors of the present invention into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in 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, lipofection, electroporation, and infection with recombinant viral vectors. Additionally, see US Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

[0173] It is understood that, in addition to containing the necessary elements for the transcription and translation of the inserted coding sequence (encoding a polypeptide), the expression constructs of the invention may also contain sequences engineered to optimize the stability, production, purification, yield, or activity of the expressed polypeptide.

[0174] Another aspect provides a pharmaceutical agent produced by the method of the invention.

[0175] Another aspect provides a pharmaceutical composition comprising an agent produced by the method of the invention and a pharmaceutically acceptable carrier, excipient, or adjuvant.

[0176] 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 1000 nanometers (nm) refers to a length of 1000 nm ± 100 nm.

[0177] It should be noted 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, a reference to a "polynucleotide" includes a plurality of such polynucleotides, and a reference to a "polypeptide" includes a reference to one or more polypeptides and equivalents thereof known to those skilled in the art. It should further be noted that the claims may be drafted to exclude any element. Accordingly, this statement is intended to serve as a guide for using exclusive terminology, such as "sole," "solely," and the like, in connection with reciting claim elements or using "negative" limitations.

[0178] In instances where a convention similar to "at least one of A, B, and C, etc." is used, such construction is generally intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having A, B, and at least one of C" would include, but be limited to, systems having A only, B only, C only, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will further be understood by those of ordinary skill in the art that virtually any disjunctive 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 terms. For example, the phrase "A or B" would be understood to include the possibilities of "A" or "B" or "A and B."

[0179] It will be understood that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments related to the present invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination were individually and explicitly disclosed. Furthermore, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if all such subcombinations were individually and explicitly disclosed herein.

[0180] Additional objects, advantages, and novel features of the present invention will become apparent to those skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as described hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0181] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. [Example]

[0182] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are fully explained in the literature. See, for example, "Molecular Cloning: A Laboratory Manual" by Sambrook et al. (1989); "Current Protocols in Molecular Biology" Volumes I-III by 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. New York (1998), methodologies 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 by reference. Other general references are found throughout this specification.

[0183] material and method Antibodies - Commercially available mouse monoclonal anti-CD28 clone #CD28.2 (Biolegend, catalog number 302902) and FITC-conjugated (Biolegend, catalog number 302906). Goat polyclonal anti-CD28 (R&D systems, catalog number AF-342-PB). FITC-conjugated anti-human PD-L1 (BD bioscience, catalog number 558065). APC-conjugated anti-human PD-L2 (Biolegend, catalog number 345508). PE-conjugated anti-human IDO (R&D systems, catalog number IC6030P). Goat anti-mouse IgG Alexa Fluor 647 (Biolegend, catalog number 405322). Donkey anti-human IgG (H+L) Alexa Fluor 647 (Jackson immune research, catalog number 709-605-149). Goat anti-mouse IgG HRP (Jackson immune research, catalog number 115-035-071), anti-human CD3 clone OKT3 (Biolegend, catalog number 317304), anti-human PD-1 pembrolizumab (MK-3475), human IgG (Sigma, catalog number I4506).

[0184] Isolation of VHHs targeting the stalk region of the human CD28 receptor: Using the genetic code of peripheral blood B cells derived from naive, non-immunized llamas, a phage library consisting of particles expressing individual VHHs as fusion proteins with a C-terminal His6-Myc tag was constructed. The naive library was used to select nanobodies capable of binding to the stalk region of human CD28. Screening was performed using biotinylated recombinant CD28-Fc chimeric or oxidized dimeric peptides with the C-terminal biotin-tagged sequence "HVKGKHLCPSPLFPGPSKP (SEQ ID NO: 10)." Each antigen was bound to streptavidin magnetic beads blocked with nonfat milk. In-solution selection of phage was performed using the same antigen across three consecutive selection rounds, varying the input amount of phage and antigen concentration. Blocked beads without antigen were used as a control. Bound phage were eluted with trypsin for 20 minutes. The enrichment ratio during in-solution selection was calculated as the ratio between the number of phage eluted from CD28 antigen selection conditions and the number of phage eluted from no antigen selection conditions. 279 individual phage monoclones from the selected output were validated for antigen binding by ELISA in either phage or periplasmic formats and characterized for binding to membrane CD28 by flow cytometry. 72 clones showed specific binding to the stalk region peptide in the periplasmic format, 22 were proven to have unique CDR sequences, and only six were found to belong to distinct CDR3 families. Six VHHs with C-terminal His tags were produced as recombinant proteins in CHO cells and evaluated for anti-shedding activity and cell binding. Transfection: CD28wt (encoding the 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 G418-containing medium.

[0185] ELISA - Commercially available ELISA kits were used to quantify the amounts of human interferon-γ (Biolegend, catalog number 430103), human interleukin-2 (Biolegend, catalog number 431802), human interleukin-6 (Biolegend, catalog number 430502), human interleukin-10 (Biolegend, catalog number 430603), human tumor growth factor beta 1 (Biolegend, catalog number 436708), human interleukin beta 1 (Biolegend, catalog number 437004), and human CD28 (R&D system, catalog number DY342). Cell proliferation and viability (MTT assay) were performed according to the manufacturer's instructions (Roche, catalog number 11465007001). Kynurenine (IDO activity) ELISA kit was performed according to the manufacturer's instructions (ImmuSmol, catalog number BA E-2200).

[0186] 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. Serial dilutions (0.2–5 μg / mL) of VHH clones were performed. Bound VHHs were detected using an anti-His tag-HRP-conjugated antibody and developed with TMB.

[0187] Cytokine multiplex - Simultaneous assessment of several cytokines was performed on the Magpix system (Millipore) using ProcartaPLex (Invitrogen, Cat. No. PPX-07-MXXGPY2).

[0188] Flow cytometry - Generally, cells were kept on ice at all steps. Before staining, 5 × 10 cells were collected. 5Cells were blocked with 50 μg / mL human IgG (Sigma, catalog no. 14506) in FACS buffer (PBS containing 0.1% BSA) for 15 minutes. Antibodies were used at the manufacturer's recommended concentrations and incubated in the dark for 30 minutes. Incubations were performed in a 100 μL volume 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 Gallios Flow Cytometry Acquisition Software.

[0189] Cell lines and human immune cell isolation—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, catalog no. 850494). CD3 cells were isolated from fresh blood samples of healthy donors by negative selection using the RossetteSEP™ Human T Cell Enrichment Kit (STEMCELL, catalog no. 15061). CD4 cells were isolated from fresh blood samples of healthy donors by negative selection using the EasySep™ Human CD4 T Cell Enrichment Kit (STEMCELL, catalog no. 19059). Monocytes were isolated from fresh blood samples of healthy donors by negative selection using the EasySep™ Human Monocyte Enrichment Kit (STEMCELL, catalog no. 17952). All cells were grown in complete RPMI-1640 medium supplemented with 10% HI-FCS and pen / strep mixture.

[0190] CD86-blocked FACS-treated 0.5 x 10 cells stably transfected with human CD28 6HEK293 cells were incubated with 2 μg / mL CD86-Fc (R&D Systems, Cat. No. 141-B2) in the presence or absence of anti-CD28 antibody (CD28.2, 10 μg / mL) or VHH clone (30 μg / mL) for 30 minutes at room temperature. Cells were washed and harvested for secondary binding using fluorophore-conjugated anti-human heavy and light chain antibodies at a 1:5000 dilution on ice for 20 minutes.

[0191] Dendritic cell differentiation - Monocytes were cultured at 1 x 10 in RPMI medium with refreshed growth factors on days 3 and 6. 6 Immature dendritic cells (iDCs) were cultured at a density of 100 / mL. Immature dendritic cells (iDCs) were induced with 50 ng / mL GM-CSF and 20 ng / mL IL-4 for 6 days. When necessary, iDCs were further differentiated into mature dendritic cells by adding 100 ng / mL LPS for 48 hours. The resulting cell populations were tested for the indicated phenotypes by FACS analysis of relevant markers and by analysis of secretion of characteristic cytokines.

[0192] Metalloproteinases - Commercially available recombinant human metalloproteinase MMP-2 was used from Anaspec (catalog number AS-72005) or R&D Systems (catalog number 902-MP). Commercially available recombinant human metalloproteinase MMP-13 was purchased from R&D Systems (catalog number 511-MM). Pro-MMP2 and Pro-MMP-13 were activated with 1 mM p-aminophenylmercuric acetate (APMA) at 37°C for 1–2 h according to the manufacturer's protocol.

[0193] Protease inhibitors—Protease inhibitors were added at the indicated concentrations at the beginning of each experiment. For 1-week assays, additional inhibitors were added at the final concentrations after 3 days. The protease inhibitors used were TAPI-1 (Cayman, catalog no. 18505), GM6001 (Santa Cruz, catalog no. SC-203979), TMI-1 (Sigma, catalog no. PZ0336), and GI254023X (Sigma, catalog no. SML0789). When referred to, the protease cocktail was an equimolar mixture of TAPI-1 and GM6001.

[0194] A synthetic substrate peptide with the final structure "DYKDDDDKGGGGGHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 41)-biotin" was designed to contain the amino acid sequence of the human CD28 stalk region (His134-Pro152) between an N-terminal cMyc tag, followed by a five-glycine sequence, and a C-terminal biotin linkage. This peptide was custom synthesized by Gencust Europe. A cysteine residue at position 141 was used to generate a dimeric peptide via a disulfide bond. A CD28 stalk region peptide with a mutation at the cleavage site, where leucine at position 145 was replaced with lysine, was similarly synthesized with the final structure "DYKDDDDKGGGGGHVKGKHLCPSPKFPGPSKP (SEQ ID NO: 42)-biotin."

[0195] In vitro cleavage assay: 50 ng of purified recombinant MMP-2 or MMP-13 was incubated with 0.125 μM of dimeric c-Myc-tagged and biotinylated substrate peptide for 5 h in the presence or absence of an MMP inhibitor (TMI-1, 50 nM), various concentrations (0.4–10 μg / mL) of M9 Fab or the indicated VHH clones. The assay was performed in 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35, pH 7.5. After 5 h, the cleavage reaction mixture was diluted to a final peptide concentration of 1 nM and loaded onto a neutravidin plate to allow binding to the peptide. After a 1-h incubation at room temperature, the plate was washed, and uncleaved peptide was detected using an anti-cMyc antibody conjugated to HRP.

[0196] PHA activation of CD4 T cells or Jurkat T cell lines for generation of soluble CD28 - 1 x 10 5 Jurkat cells or CD4 T cells were incubated with the indicated concentrations of phytohemagglutinin (Sigma, catalog no. L8902) and various protease inhibitors for an additional 5 days (Jurkat) or 7 days (CD4 T cells).

[0197] SEB or CMV activation of PBMCs to generate soluble CD28 - 0.3 x 10 in a 48-well plate 6 PBMCs were stimulated with 0.5 ng / mL SEB (Sigma, Cat. No. S4881) with or without various protease inhibitors at the indicated concentrations for 5–7 days at 37°C. Alternatively, 0.1 × 10 cells were used in a 96-well plate format assay. 6 PBMCs were stimulated with 0.5 ng / mL SEB. For CMV stimulation, 0.5 × 10 PBMCs were plated in 96-well plates with or without various protease inhibitors at the indicated concentrations. 6PBMCs were stimulated with 0.5 μg / mL CMV peptivator (Milteny Biotec, catalog no. 130-093-435) for 2–5 days at 37°C. For sequential shedding experiments, PBMCs were stimulated with SEB or CMV in 24-well plates for 24 hours, then cells were harvested, washed three times with RPMI without stimuli, and replated in 96-well plates. Samples were harvested at the indicated times and kept frozen until assayed for soluble CD28.

[0198] For the cell assay to assess the anti-shedding activity of VHHs - SEB activation of PBMCs, 0.1 x 10 6 PBMCs were stimulated with 2 ng / mL SEB (Sigma, Cat. No. S4881) with or without various treatments at the indicated concentrations for 5–7 days at 37°C. PHA-activated T cells were cultured in 96-well plates at 0.1 × 10 cells with or without various treatments at the indicated concentrations. 6 CD4 T cells were stimulated with the indicated concentrations of phytohemagglutinin (Sigma, Cat. No. L8902) and 200 IU / mL IL-2 (Proleukine) for 5–7 days at 37°C. For the HEK spontaneous CD28 shedding assay, 0.1 × 10 cells were cultured in a 96-well plate. 5 HEK cells were incubated for 48 hours at 37°C with or without various treatments at the indicated concentrations.

[0199] Mixed lymphocyte reaction - 1 × 10 5 Immature DCs, 5 × 10 5 were mixed with isolated autologous CD3 T cells for 6 days.

[0200] SEB or CMV stimulation assay with ectopic recombinant human CD28, human CTLA-4, and human CD80. For CMV stimulation, 0.5 x 10 cells were cultured in a 96-well plate. 6PBMCs (from healthy or cancer patient donors) were stimulated with 0.5 μg / mL CMV peptivator (Milteny Biotec, Cat. No. 130-093-435) with or without the indicated concentrations of recombinant human CD28 (R&D systems, Cat. No. 342-CD), human CTLA-4 (R&D systems, Cat. No. 434-CT), or human CD80 (R&D systems, Cat. No. 140-B1) at 37°C for 2–5 days. In the SEB setting, 1 × 10 5 PBMCs were cultured for 72 hours at a concentration of 0.5 ng / mL Staphylococcal enterotoxin B (SEB) (Sigma, catalog no. S4881) in the presence of the indicated concentrations of recombinant (rec.) human CD28. Where indicated, anti-PD1 or human IgG was added to a final concentration of 5 μg / mL.

[0201] Autologous monocyte CD3 MLR-0.5 × 10 6 0.5 x 10 T cells from the same CMV-reactive donor 5 The cells were mixed with monocytes and stimulated with 0.5 μg / mL of CMV peptivator at 37°C for 6 days with or without the indicated concentrations of treatment.

[0202] Stimulation of monocytes with recombinant human CD28 - 1.5 x 10 6 Monocytes were seeded in 24-well plates in RPMI medium containing 100-100 U / ml IFNγ (R&D Systems, Cat. No. 285-IF) in the presence of the indicated concentrations of recombinant human CD28 for 48 h. The resulting cell populations were tested for the indicated phenotypes by FACS analysis of relevant markers (IDO, PD-L1, and PD-L2) and by analysis of secretion of a characteristic cytokine (IL-6).

[0203] T cell stimulation with OKT3 - 0.1 × 10 6Isolated CD3 T cells (from healthy donors) were stimulated with the indicated amount of anti-CD3 clone OKT3 at 37°C for 48–72 h. Soluble recombinant human CD80-Fc (2 μg / mL, R&D Systems) was added at the start of the stimulation. Soluble antibodies or VHHs against CD28 were added at the indicated concentrations.

[0204] Co-culture of SCC-25 cancer cell line with monocytes in a transwell-based assay - 4 × 10 with or without the indicated treatments for 4 days in serum-free starvation medium 4 1 x 10 SCC-25 cells were seeded in the bottom of a 24-well plate. 5 Monocytes were seeded onto cell culture inserts (Millipore, catalog number MCHT241148).

[0205] Detection of soluble human CD28 in cancer patient plasma - Twenty frozen plasma samples from each of 10 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. High sCD28 samples were reanalyzed at the appropriate dilution.

[0206] Direct CD28 EIA - Corning high-binding plates or equivalent were used for screening unless otherwise noted. Each well was coated with 200–300 ng of human CD28-Ig chimera (R&D, catalog no. 342-CD), mouse CD28-Ig chimera (R&D, catalog no. 483-CD), or a BSA-conjugated dimeric peptide consisting of the CD28 stalk region amino acid sequence (Gly137–Pro152). Plates were blocked with 5% milk or 1% casein in PBS for 1 h at room temperature (RT). Plates were washed three times with PBST and incubated with the investigated antibody, followed by goat anti-mouse HRP Fc-specific detection at a dilution of 1:5000. Positive controls were mouse anti-human CD28 clone 28.2 or mouse serum from immunized mice. Hybridoma supernatant cultures were screened undiluted.

[0207] Antibody Sequencing. Antibodies were provided to Rapid Novor for amino acid sequencing. Sequencing was performed using standard methods, which briefly included enzymatic digestion with six enzymes (pepsin, trypsin, chymotrypsin, elastase, LysC, and AspN) followed by LC-MS analysis. Digestion was performed using disulfide reduction and alkylation. LC-MS / MS analysis was performed using a Thermo-Fisher Q-exactive mass spectrometer. For both the heavy and light chains of each antibody, 100% of the amino acid residues were covered with at least five peptide scans, with significant supporting fragment ions. CDRs were determined using the Chothia scheme.

[0208] Example 1: Human CD28 undergoes proteolytic shedding during chronic stimulation Soluble CD28 (sCD28) was detected by ELISA in chronically stimulated human PBMC cultures (Figure 1, top graph). This phenomenon was evident regardless of the nature of the stimulator, either artificial (SEB) or physiological (CMV), demonstrating the robustness of the phenomenon. Treatment with TAPI-1 and GM6001 (broad-spectrum MMP and ADAM17 inhibitors) reduced the amount of sCD28 in a dose-dependent manner, suggesting the origin of soluble CD28 is shedding of the membrane form (Figure 1, top graph). The cellular source of shed CD28 is T cells, as seen in Figure 2. Chronic stimulation of a Jurkat T cell line or human CD4 T cells from peripheral blood of healthy donors with PHA produces sCD28 in a dose-dependent manner (Figure 2, top graph). Treatment with TAPI-1 and GM6001 reduced the amount of sCD28 in a dose-dependent manner at each PHA concentration (Fig. 2, upper graph) and at a fixed PHA concentration (Fig. 2, lower graph).

[0209] Treatment with GI254023X, a highly specific ADAM-10 inhibitor, resulted in a nearly complete inhibition of sCD28 release from activated immune cells in a dose-dependent manner (Figure 3A, lower panel). Similar results were observed with the ADAM-17-specific inhibitor TMI-1 (Figure 3B, lower panel). Immune cell viability was monitored by MTT assay to confirm the metabolic activity of cells in culture. Results showed no significant difference between treatments with and without the ADAM inhibitor, suggesting that low sCD28 levels were due to blockade of protease activity and not an artifact of protease inhibitor-induced cell death (Figure 3A-B, upper panel).

[0210] The generation of sCD28 was also validated in a more physiological system. First, CD4 T cells were utilized, mimicking the physiological stimulation of T cells with isolated autologous dendritic cells and antigen-presenting cells. The increase in sCD28 was evident when the two cell populations were mixed and even more pronounced when CMV was added to the culture (Figure 4A). This indicates that upon chronic stimulation, the human CD28 protein undergoes a proteolytic shedding process.

[0211] Next, human PBMCs were stimulated with CMV peptide (Figure 4B) or SEB (Figure 4C) for 24 hours. The cells were then washed to remove the stimulator and re-seeded for various periods without stimulation. The presence of sCD28 in the culture medium was then examined. The accumulation of sCD28 was clearly visible over time. Furthermore, as seen in Figure 4D, the accumulation depended on the activity of ADAM-10 and ADAM-17. Addition of specific inhibitors at different concentrations after SEB stimulation reduced the amount of sCD28 quantified after 120 hours. Because CD28 shedding occurs after primary T cell activation and does not necessarily require constant or repeated stimulation, this study may explain the presence of large amounts of soluble CD28 in patient blood.

[0212] Example 2: Soluble human CD28 has immunosuppressive activity As seen in Figure 1 (bottom graph), reducing sCD28 levels using a protease inhibitor cocktail directly correlated with increased T cell activation, as evidenced by levels of secreted IFNγ, suggesting that sCD28 has an immunosuppressive function. Increasing the concentration of the protease inhibitor cocktail led to decreased sCD28 levels in the cell culture medium, and these decreases in sCD28 levels were inversely correlated with increased levels of secreted IFNγ. To further investigate immunosuppression by sCD28, recombinant human CD28 lacking the transmembrane and cytoplasmic domains was added to the culture medium of human PBMCs stimulated with CMV. This resulted in a dose-dependent inhibition of IFNγ secretion (Figure 5). This immunosuppressive effect was observed across different human PBMC donors, confirming the robustness of this signaling axis blocked by sCD28.

[0213] In parallel, elevated secretion of interleukin-6 (Figure 6 and Figure 7A) and interleukin-10 (Figure 7A) was evident. These cytokines have been reported to indicate a skewing of the immune system toward a type 2 immune response, which may support cancer growth and angiogenesis through suppression of immune effector activity (IL-10) and STAT-3 signaling (IL-6). Furthermore, a comparison was made with soluble CTLA-4 (mimicking abatacept—a registered therapeutic agent for autoimmune disorders), revealing an overall similar effect on the immune system in terms of cytokine secretion profile (Figure 7A).

[0214] Next, human PBMCs were stimulated with SEB (1 ng / mL) in the presence or absence of recombinant human CD28. Human IgG was used as a control. Lymphocyte clustering, a hallmark of immune activation, was monitored using IncuCyte® S3 live cells in photographs taken every 12 hours. As seen in Figure 7C, SEB had essentially no effect on lymphocytes in the presence of recombinant human sCD28. It is well established that antigen-presenting cells (APCs) cluster with each other and with other cell types during in vitro immune responses, and that clustering is essential for antigen-specific activation of resting lymphocytes. Soluble CD28 appears to reduce the amount and size of cluster formation during the SEB immune response, implying that it inhibits the first step in T cell-specific activation by APCs.

[0215] Similar results were observed when isolated autologous monocytes and CD3 T cells were cocultured in a mixed lymphocyte reaction (MLR). The mixed cells were stimulated for 5 days with CMV peptide (0.5 μg / mL) with or without increasing concentrations of recombinant human sCD28. Again, sCD28 was found to inhibit IFNγ secretion while simultaneously increasing IL-1B, TGFβ, and IL-10 secretion (Figure 7B).

[0216] sCD28 had a similar immunosuppressive effect on monocytes. The enzyme indoleamine 2,3-dioxygenase (IDO) is involved in immunoregulation due to its ability to catabolize the essential amino acid tryptophan. It is also expressed in various immune cells and many cancer cells. Tryptophan deficiency inhibits T lymphocyte maturation and proliferation, while kynurenine, the end product of tryptophan catabolism, is also known as an immunosuppressive metabolite that promotes immune tolerance in various physiological and pathophysiological conditions. To test the effect of sCD28 on IDO, isolated human monocytes were stimulated with IFNγ (1000 U / mL) for 48 hours in the presence of control human IgG or recombinant human CD28 (10 μg / mL). After incubation, monocytes were intracellularly stained for human IDO (Figure 7E). To facilitate intracellular staining, cells were fixed and permeabilized with BD Cytofix / Cytoperm buffer kit. Culture media from various treatments were assessed for IDO activity using an ImmuSmol specific kynurenine ELISA kit (Fig. 7D). sCD28 strongly enhanced IDO expression in monocytes.

[0217] Moreover, surprisingly, sCD28 was 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 proinflammatory cytokine secretion (IFNγ and IL-2), but the presence of sCD28 completely abolished this immune-activating effect (Figure 8A).

[0218] Similar results were observed in the MLR setting. MLR was performed as before, with or without sCD28 and with or 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.

[0219] To elucidate the mechanism by which sCD28 inhibits the pro-activating effects of anti-PD-1 therapy, we examined the expression of PD-1 ligands on immune cells in the presence of sCD28. Isolated human monocytes were stimulated for 48 hours with IFNγ (1000 U / mL) or recombinant human CD28 (10 μg / mL) in the presence of control human IgG (10 μg / mL). After incubation, monocytes were stained for PD-L1 (Figure 8C, left) and PD-L2 (Figure 8C, right). Both ligands were upregulated on monocytes cultured with sCD28, suggesting one possible way that sCD28 circumvents the effects of anti-PD-1 immunotherapy.

[0220] Example 3: Soluble human CD28 is found in the plasma of cancer patients sCD28 levels in cancer were found only in a small number of breast cancer patients and were only slightly above those observed in healthy individuals (Isitmangil, G., In vivo, 2016). The authors suggest that sCD28 could be used as a marker for breast cancer, but no functional relationship was suggested. Now that we know that soluble CD28 may indeed enhance the immune system's ability to evade cancer, we conducted a study of 220 samples covering 10 different cancer indications and 20 samples from healthy donors. The study found high sCD28 levels in several cancers, sometimes orders of magnitude higher than those seen in healthy controls or even breast cancer patients (Figure 9A). Indeed, when compared to sCD28 levels found in some melanoma, colorectal cancer, ovarian cancer, NSCLC, and head and neck cancer patients, levels in breast cancer patients appear to be comparable to those in healthy individuals.

[0221] To further elucidate the role of sCD28 in cancer, PBMCs were isolated from cancer patients with different indications. Cells were stimulated with SEB (5 ng / mL) for 3 days alone, with MK-3475, with recombinant human sCD28, or with a combination of both molecules. The concentration of human IFNγ in supernatants from cells from all donors was significantly reduced in the presence of sCD28, even in the presence of MK-3475 (Figure 9B). Indeed, the effect of MK-3475 was absent in the presence of sCD28.

[0222] Next, cells of the head and neck cancer cell line SCC-25 were incubated alone or with monocytes in a transwell assay. Treatment of SCC-25 cells growing alone with IL-6 as a positive control indeed increased cell proliferation, as measured by MTT (Figure 9C, top) and % confluence (Figure 9C, bottom). Growth of cancer cells in the presence of monocytes also increased proliferation, but by far the greatest increase was observed when sCD28 was included in the coculture. This data further supports the pro-cancer effects of sCD28.

[0223] Example 4: sCD28 inhibits the efficacy of CD80-Fc CD80, along with CD86, is one of the two major ligands for mCD28. The extracellular domain of CD80 fused to the Fc portion has been used as an immunostimulatory molecule and is being investigated as a cancer therapeutic. To examine the effect of sCD28 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 of soluble recombinant human CD80-Fc. As expected, CD80-Fc increased IFNγ secretion. However, the addition of sCD28 counteracted the secondary activating effect of CD80-Fc (Figure 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 (Figure 10B).

[0224] Example 5: Effect of sCD28 on cancer in vivo Because mice do not cleave mCD28, the effects of sCD28 cannot be easily tested in mouse models. The closest option is to administer recombinant sCD28 to mice to mimic the situation of elevated sCD28 levels. This was investigated in the H22 syngeneic mouse model. Balb / c fully immunocompetent mice were allografted with H22 hepatocellular carcinoma cells. The cells proliferated in fully immunocompetent mice, and the addition of anti-PD-1 therapy almost completely abolished tumor growth (Figure 11A). Addition of recombinant human sCD28 almost completely abolished the effects of anti-PD-1 therapy in two mice (Figure 11B). This suggests that, in some subjects, increased sCD28 levels may have a profoundly detrimental effect on cancer progression.

[0225] Example 6: Characterization of anti-shedding antibody-based drugs The discovery that human CD28 undergoes proteolytic processing by ADAM10 and ADAM17 prompted an examination of its polypeptide sequence for candidate regions potentially susceptible to proteolytic shedding. Studies suggest that ADAM10 and ADAM17 prefer leucine, valine, and aromatic residues at the P1' position. The most attractive sequence region of human CD28 is the stalk section spanning from histidine 134 to proline 152 (SEQ ID NO: 10: HVKGKHLCPSPLFPGPSKP), which connects the globular IgV domain to the transmembrane region. This region contains a total of three leucine and valine residues, as well as a phenylalanine residue, and is predicted to lack secondary structure elements that may hinder protease access. Notably, the stalk region also contains cysteine 141, which forms a disulfide bond that promotes CD28 homodimerization. To generate antibodies or antibody fragments that specifically bind to the CD28 stalk region and potentially block access of different proteases to shed CD28 while avoiding degradation of CD28 oligomer structure and function, we immunized CD1 mice with a dimeric peptide mimicking the CD28 stalk region. The peptide sequence used for immunization was SEQ ID NO: 29, GKHLCPSPLFPGPSKPK. Using hydrazide chemistry, a C-terminal lysine was added to provide a free amino group that allowed for conjugation of KLH or BSA. Conjugation was performed between the hydrazide-terminated CD28 peptide and S-4FB-modified BSA, which generates a free aldehyde for site-specific conjugation. Dimerization was confirmed by running the peptide on a non-denaturing gel.

[0226] An antibody with high binding affinity for recombinant human CD28 was found, as measured by direct CD28 EIA. This antibody is designated M9, and its sequence is provided above. Using serial dilutions of antibody M9, specific binding to recombinant human sCD28 and the stalk region peptide was confirmed (Figure 12A). Interestingly, the antibody was able to detect recombinant human sCD28, but was unable to detect sCD28 that had actually been shed from immune cells (Figure 12B). This strongly suggests that the antibody binds at the cleavage site and that the deisotopes it binds are incomplete in the cleaved form.

[0227] Next, we investigated the ability of antibodies to bind to mCD28 on the cell surface. To reduce shedding of sCD28 from cells, the antibody would need to actually bind to the membrane form of the protein, not just the recombinant protein in solution. HEK293 cells overexpressing human full-length CD28 were analyzed. Because mouse CD28 does not appear to be cleaved into a soluble form (activated mouse splenocytes do not appear to produce sCD28), the human protein had to be investigated. Cells were analyzed by flow cytometry using M9 and CD28.2 antibodies as positive controls. Surprisingly, M9 did not appear to bind to surface mCD28 (Figure 12C). This may be due to steric hindrance and limited access to the stalk region when adjacent to the membrane.

[0228] Example 7: Single domain antibodies inhibit sCD28 shedding from the cell surface We designed small drugs that can bind to mCD28 on the surface of cells and block sCD28 shedding. Full-length antibodies are approximately 150 kDa in size, but Fab fragments derived from antibodies are approximately 50 kDa in size, while single-chain antibodies (also called single-chain variable fragments, scFv) are approximately 25 kDa in size, and single-domain antibodies (also called VHH antibodies, scFv, and DARPins) are only 12-15 kDa in size.

[0229] Single-domain antibodies were isolated using a phage library of VHHs derived from naive llamas. The library was composed of VHH sequences collected from naive, non-immunized llamas; B cells were extracted and the entire repertoire of available VHH CDRs was sequenced. These CDRs were then packaged into phage to generate the library. Using ELISA and flow cytometry, the library was screened against recombinant CD28 extracellular domain and dimeric stalk region peptides to identify antibodies that specifically bind to the stalk region of human CD28. The VHH sequences that were found to specifically bind to the stalk region of human CD28 were: EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVTVSSAAAHHHHHH (SEQ ID NO: 45, clone 2A1), EVQLVESGGGLVQAGGSLRLSCAASGSLFSINAMAWYRQAPGKQRELVAAITSSGS The VHHs were produced as recombinant proteins in CHO cells and then evaluated for cell binding and anti-shedding activity as described below. A C-terminal His tag was used for purification and linked via the triple alanine repeat. Table 1 provides the CDRs of the three clones investigated. [Table 1]

[0230] Binding of VHH clones to the human CD28 stalk region sequence was first confirmed by ELISA using serial dilutions of the VHH clones (Figure 13). Binding to human membrane CD28 at the cellular level was confirmed by FACS analysis using labeled VHH clones and HEK cells overexpressing CD28 (Figure 14). Membrane CD28 binding indicates access to the CD28 proximal membrane region. Previous experiments showed that full-length antibodies capable of binding to CD28 stalk region peptides failed to bind to the CD28 stalk region on cells, indicating that drug size is important for accessing this region. Notably, VHH clones were unable to bind to a human CD28 stalk region sequence containing an LK substitution at amino acid residue 145, located within the MMP cleavage site (Figure 23).

[0231] Anti-shedding activity was confirmed at both the peptide and cellular levels. Using ELISA technology, we confirmed that the VHH clones blocked cleavage of the human CD28 stalk region by MMP-2 (Figure 17) and MMP-13 (Figure 22) by detecting an intact human CD28 stalk region dimer peptide. On the other hand, although M9 Fab demonstrated the ability to block cleavage of the CD28 stalk region peptide by MMP-2 as described above, it was unable to bind to the CD28 stalk region on cells and inhibit CD28 shedding from the cell membrane. At the cellular level, we confirmed the efficacy of the VHH clones in inhibiting sCD28 shedding by measuring human sCD28 levels in the supernatants of human CD28-overexpressing HEK cells (Figure 18), isolated CD4 T cells activated with PHA and IL-2 (Figure 19), and PBMCs activated with superantigen (Figure 20). As expected, M9 Fab did not reduce sCD28 levels in the supernatant, further highlighting the importance of the size and architecture of the blocking agent to its ability to actually block shedding.

[0232] Importantly, the VHH clones were found to not impair human CD28 function. Using flow cytometry, we found that the VHH clones did not alter the magnitude of CD86 binding to membrane CD28 (Figure 15). Using a standard sandwich ELISA, we demonstrated that the VHH clones did not stimulate CD28, as measured by the secretion levels of the inflammatory cytokine interferon gamma (Figure 16). The activating antibody CD28.2 was used as a positive control. Similarly, using a standard sandwich ELISA, we demonstrated that the VHH clones did not antagonize CD80-Fc stimulation via CD28, as measured by the secretion levels of the cytokine IL-2 (Figure 21).

[0233] Example 8: Design of other small drugs to inhibit sCD28 shedding from the cell surface Fab fragments are generated using commercially available kits or services. The CDR region of antibody M9, which has been shown to bind to the appropriate deoxyribonucleotides, is used for Fab generation. The efficacy of the resulting Fab fragments is first tested by binding assays to recombinant human CD28 and dimeric stalk region peptides to confirm that this binding is maintained. Binding to surface mCD28 is assayed by FACS on mouse cells expressing human CD28 and human immune cells. 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 cultured in the presence and absence of Fab fragments. Fab fragments with shedding-inhibitory activity are assayed for their effect on CD28 signaling. Agonism is first tested by assaying the ability of the Fab fragments to induce the secretion of inflammatory cytokines (e.g., interferon-γ) from T cells. Second, the antagonistic properties of the Fab fragments are tested using their ability to block binding of CD80-Fc (agonist).

[0234] Single-chain antibody production using the M9 CDRs is performed by standard methods using commercial services or by inserting the CDRs into an scFv framework. Purification is performed and the resulting antibodies are evaluated using the same assays described for the Fab fragments.

[0235] Single domain antibodies are produced by one of two strategies: 1) Naive library - using a phage library of VHHs derived from naive bruma. The library is composed of VHH sequences collected from naive bruma spleens, i.e., B cells are extracted, and the entire available repertoire of VHH CDRs is sequenced. These CDRs are packaged on phage to generate a library. The library is screened against recombinant CD28 extracellular domain and dimeric stalk region peptides to identify antibodies that specifically bind to sCD28. 2) Immune library - llamas or other camelids or sharks are immunized with cells overexpressing CD28. After cell immunization, the spleen is extracted and the available repertoire of VHH CDRs is sequenced. Hybridomas are generated from the extracted splenic B cells. The resulting antibodies are packaged on phage to generate a library, which is then screened against recombinant CD28 extracellular domain and dimeric stalk region peptides to identify antibodies that specifically bind to sCD28. Single domain antibodies with specific binding are assessed for blocking of shedding and agonism / antagonism, as is done for Fab fragments and single chain antibodies.

[0236] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

Claims

Claim 1: A single domain antibody that binds to the stalk region of membrane CD28 (mCD28), said single domain antibody being a camelid antibody, said camelid antibody comprising three CDRs: 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); or 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 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: 39 (AITSGGSTNYADSVKG), and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 40 (DLYGEDYWI); The stalk region has the amino acid sequence HVKGKHLCPSPLFGPSKP (SEQ ID NO: 10), Single domain antibodies.

2. The camelid antibody, a. EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVTVSS (SEQ ID NO: 30); b. EVQLVESGGGLVQAGGSLRLSCAASGSLFSINAMAWYRQAPGKQRELVAAITSSGSTNYANSVKGRFTVSRDNAKNTMYLQMNSLKPEDTAVYYCVVDEYGSDYWIWGQGTQVTVSS (SEQ ID NO: 31), and c. QVQLVESGGGLVQAGGSLRLSCAASGSIFSINAMGWYRQAPGKQRERVAAITSGGSTNYADSVKGRFTISRDNAKNTVYLQMNNLEPRDAGVYYCVVDLYGEDYWIWGQGTQVTVSS (SEQ ID NO: 32) 2. The single domain antibody of claim 1, comprising a sequence selected from the group consisting of:

3. 3. A single domain antibody according to claim 1 or 2 which is not a CD28 agonist.

4. A single domain antibody according to any one of claims 1 to 3 which is not a CD28 antagonist.

5. The single domain antibody of any one of claims 1 to 4, which does not degrade mCD28 or inhibit mCD28-mediated immune cell activation.

6. The single domain antibody of any one of claims 1 to 5, wherein the single domain antibody blocks cleavage of the stalk region by MMP-2, MMP-13, or a combination thereof.

7. 7. A single domain antibody according to any one of claims 1 to 6 for use in reducing soluble CD28 (sCD28) levels in a subject in need thereof.

8. 7. A single domain antibody according to any one of claims 1 to 6 for use in treating and / or preventing cancer in a subject in need thereof.

9. 7. The single domain antibody of any one of claims 1 to 6 for use in improving PD-1 and / or PD-L1 based immunotherapy in a subject in need thereof.

10. 10. The single domain antibody for use according to claim 7 or 9, wherein the subject is suffering from cancer.

11. 11. The single domain antibody for use according to claim 8 or 10, 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.

12. 12. The single domain antibody for use according to claim 11, wherein the cancer is selected from melanoma, head and neck cancer, non-small cell lung cancer, ovarian cancer and colorectal cancer.

13. A single domain antibody for use according to any one of claims 7 to 12, wherein said use does not degrade mCD28 or reduce mCD28-mediated immune cell activation.

14. A single domain antibody for use according to any one of claims 7 to 13, wherein the blood of the subject prior to said use contains at least 5 ng / ml of sCD28.

15. 1. A method for producing a single domain antibody that inhibits proteolytic cleavage of mCD28 on the cell surface, comprising: a. i. Obtaining a single domain antibody that binds to the stalk region of the CD28 extracellular domain, said stalk region consisting of the amino acid sequence HVKGKHLCPSPLFGPSKP (SEQ ID NO: 10); ii. testing the binding of the obtained single domain antibodies to mCD28 on the cell surface; iii. Selecting a single domain antibody that binds to cell surface mCD28; iv. testing the ability of the selected single domain antibodies to block cleavage of mCD28 on the cell surface by proteases; and v. selecting a single domain antibody that binds to the stalk region of mCD28 on the cell surface and blocks cleavage of mCD28 on the cell surface by proteases; and b. Culturing a host cell comprising one or more vectors comprising a nucleic acid sequence encoding a single domain antibody, said nucleic acid sequence comprising: i. Obtaining a single domain antibody that binds to the stalk region of the CD28 extracellular domain, said stalk region consisting of the amino acid sequence HVKGKHLCPSPLFGPSKP (SEQ ID NO: 10); ii. testing the binding of the obtained single domain antibodies to mCD28 on the cell surface; iii. Selecting a single domain antibody that binds to cell surface mCD28; iv. testing the ability of the selected single domain antibodies to block cleavage of mCD28 on the cell surface by proteases; and v. selecting a single domain antibody that binds to the stalk region of mCD28 on the cell surface and blocks cleavage of mCD28 on the cell surface by proteases; a nucleic acid sequence of a single domain antibody selected by and Thereby generating single domain antibodies that inhibit proteolytic cleavage of mCD28 on the cell surface.

16. The method of claim 15, wherein the protease is selected from MMP-2 and MMP-13.

17. Obtaining said single domain antibody, a. immunizing a shark or camelid with said stalk region of the CD28 extracellular domain and collecting antibodies from the immunized organism; and b. screening a library of single domain antibodies for binding to the stalk region of the CD28 extracellular domain and selecting those that bind; 17. The method of claim 15 or 16, comprising at least one of the following:

18. The method of claim 17, wherein the stalk region of the CD28 extracellular domain is a dimer or a monomer.

19. a. harvesting said antibodies comprises extracting B cells from the spleen of said immunized shark or camelid; or b. selecting said binding single domain antibody comprises sequencing said selected single domain antibody and generating a recombinant form of said single domain antibody from said sequence; 19. The method of claim 17 or 18.

20. The method of any one of claims 15 to 19, further comprising assaying mCD28 downstream signaling in the presence of said obtained single domain antibodies and selecting at least one single domain antibody that neither substantially agonizes nor substantially antagonizes mCD28 signaling.

21. A pharmaceutical composition comprising a single domain antibody according to any one of claims 1 to 6 and a pharma- ceutically acceptable carrier, excipient or adjuvant.

22. 22. The pharmaceutical composition of claim 21 for use in treating and / or preventing cancer, improving PD-1 and / or PD-L1 based immunotherapy, or reducing levels of sCD28 in a subject in need thereof.

23. A kit comprising at least one single domain antibody according to any one of claims 1 to 6.

24. a. anti-PD-1 and / or PD-L1 immunotherapy, and b. A label indicating that the single domain antibody is for use with PD-1 and / or PD-L1 based immunotherapy; 24. The kit of claim 23, further comprising at least one of:

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