Small shedding blocker

Abstract

The name of the invention is small shedding blockers. In some embodiments, less than 100 thousand Daltons of agents are provided that bind to a membrane immune receptor on the surface of a cell and inhibit protease cleavage of the immune receptor. Also provided are methods of treating cancer and enhancing immunotherapy comprising administering the agents.

Description

[0001] This application is a divisional application. The filing date of the original application is March 12, 2020, the application number is (202080028967.4), and the title is "Small shedding blocker".

[0002] Cross-reference to related applications

[0003] This application claims the priority benefits of U.S. Provisional Patent Application No. 62 / 954,802 filed on December 30, 2019, U.S. Provisional Patent Application No. 62 / 942,240 filed on December 2, 2019, and U.S. Provisional Patent Application No. 62 / 818,351 filed on March 14, 2019, the entire contents of which are incorporated herein by reference in their entirety. Technical field

[0004] The present invention belongs to the fields of immunomodulation and immunotherapy. Background art

[0005] The adaptive immune system plays a key role in the regulation and protection against pathogens and cancer cells, mainly by coordinating the stimulation of antigen-specific helper CD4+ and cytotoxic CD8+ T cells. Persistent and continuous activation of T cells by antigen-presenting cells (APCs) involves i) the engagement of the T cell receptor (TCR) with peptides presented by major histocompatibility complex (MHC) on the APC; and ii) the co-stimulatory CD28 receptor on T cells binding to the B7-1 (CD80) and B7-2 (CD86) ligands also expressed by the APC. The biological consequences of CD28 co-stimulation are numerous and include controlling the T cell cycle, expansion, differentiation, and amplification of TCR stimulation by reducing the threshold required to achieve immune effector functions.

[0006] Unlike the activating co-stimulatory molecule CD28, the structural homolog, cytotoxic T lymphocyte-associated 4 (CTLA-4), is an inhibitory co-stimulatory receptor whose membrane expression is driven by the triggering of CD28. CTLA-4 and CD28 are both type I transmembrane proteins. Their extracellular portions consist of a group V immunoglobulin superfamily (Ig-V) domain, which is covalently linked homologously by cysteine residues located near the transmembrane region outside the IgV domain. Although similar, CTLA-4 and CD28 differ in terms of affinity and quaternary structure arrangement. CTLA-4 has been found to have a higher binding affinity for B7 molecules, and the different dimerization patterns from CD28 result in different stoichiometric bindings to the shared ligands. CD28 exhibits a monovalent binding stoichiometry, while CTLA-4 interacts in a bivalent manner. Thus, CTLA-4 binds to B7 molecules with much higher affinity and antibody avidity than CD28, and thereby downregulates T cell responses and contributes to the initiation of antigen-specific tolerance.

[0007] It has been shown that some costimulatory molecules have several physiological forms. In addition to the membrane-bound form, a soluble form expressed in naive immune cells has been described, adding to the complexity of T cell biology. The soluble form of CD28 (sCD28) has been attributed to an alternatively spliced gene product. The splicing event results in a frameshift, with the consequence that two glutamate residues are added after glycine at position 137 before translation termination. The final product lacks the entire transmembrane and cytoplasmic regions and, importantly, lacks the cysteine residue at position 141, which mediates the disulfide bond of dimeric CD28 (Magistrelli G., Biochem Biophys Res Commun, 1999). The biological function and counter-receptor binding of the monomeric soluble form of CD28 have been examined (Hebbar, M., Clin Exp Immunol, 2004) and shown to also inhibit T cell proliferation. Similarly, in the case of dimeric sCD28, a regulatory role has been proposed in inhibiting T cell function by binding to B7 molecules (Sun, Z., Centr Eur J Immunol, 2014; Hebbar, M., Clin Exp Immunol, 2004). Notably, an increased number of sCD28 molecules has been reported in the sera of patients with autoimmune disorders (Wong, C.K., Rheumatol, 2005; Hamzaoui, K., Clin Exp Rheumatol, 2005; Hebbar, M., Clin Exp Immunol, 2004; Sun, Z., Clin Immunol, 2014). The exact source of sCD28 is controversial. Using an in vitro model of T cell activation, which reflects the persistent inflammatory state of T cells in autoimmune patients, it has been shown that during T cell activation, the transcription of the alternative soluble form is inhibited and only the full-length membrane form of CD28 is evident, while the amount of sCD28 in the culture medium is elevated (Hebbar, M., Clin Exp Immunol, 2004). This phenomenon has led to the suggestion that efficient shedding of the membrane form of CD28 is the cause of the elevated soluble molecule in the serum, yet this remains to be proven. In the past, efficient shedding during T cell activation has been described as a regulatory mechanism to counter continuous activation by proteolysis of adhesion molecules.

[0008] While CTLA-4 limits the magnitude of early T cell responses, another inhibitory receptor, PD-1, suppresses T cell function in the periphery. Expression of PD-1 is elevated during T cell activation, and its known ligands are B7 family homologs: B7-H1 (PD-L1) and B7-H2 (PD-L2). These homologs are present on APCs and cancer cells and drive activated T cells into a state of cellular anergy, resulting in a diminished immune response. Accordingly, targeted therapies against the CTLA-4 and PD-1 / PD-L1 axes have shown clinical activity in multiple cancer types. Recently, studies have shown that the signaling pathway of CD28 is targeted and inhibited by PD-1 (Hui, E., Science, 2017) and concomitantly, an intact, active CD28 / B7 axis is essential for the conduct of effective PD-1 therapy (Kamphorst, A.O., Science, 2017).

[0009] However, not all patients respond to PD-1-based immunotherapy or immunotherapy in general, and those patients do often relapse. Accordingly, there is a great need for methods and molecules that can enhance the ability of a patient's immune cells to attack cancer. SUMMARY OF THE INVENTION

[0010] The present invention provides a reagent less than 100 kilodaltons that binds to membrane CD28 (mCD28) on the cell surface and inhibits proteolytic cleavage of mCD28. Also provided are methods of treating and preventing cancer and improving PD-1 / PD-L1-based immunotherapy, which comprise administering the reagent.

[0011] According to a first aspect, there is provided a reagent that binds to membrane CD28 (mCD28) on the cell surface and inhibits proteolytic cleavage of mCD28, wherein the reagent is less than 100 kilodaltons (kDa).

[0012] According to another aspect, there is provided a method of reducing the level of soluble CD28 (sCD28) in a subject in need thereof, the method comprising administering a reagent of the present invention.

[0013] 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 a reagent of the present invention.

[0014] 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 a reagent of the present invention.

[0015] According to another aspect, there is provided a method of generating a reagent that inhibits proteolytic cleavage of mCD28 on the cell surface, the method comprising at least one of the following steps:

[0016] a. Obtain a reagent that binds to the extracellular domain of CD28 or a fragment thereof, wherein the reagent is less than 100 kDa;

[0017] b. Test the binding of the obtained reagent to mCD28 on the cell surface; and

[0018] c. Select a reagent that binds to cell surface mCD28;

[0019] and

[0020] d. Culture a host cell comprising one or more vectors, wherein the one or more vectors comprise a nucleic acid sequence encoding the reagent, and wherein the nucleic acid sequence is the nucleic acid sequence of the reagent selected by:

[0021] i. Obtain a reagent that binds to the extracellular domain of CD28 or a fragment thereof, wherein the reagent is less than 100 kDa;

[0022] ii. Test the binding of the obtained reagent to mCD28 on the cell surface; and

[0023] iii. Select a reagent that binds to cell surface mCD28;

[0024] Thereby generating a reagent that inhibits protease cleavage of mCD28 on the cell surface.

[0025] According to another aspect, there is provided a reagent produced by the method of the present invention.

[0026] According to another aspect, there is provided a pharmaceutical composition comprising the reagent of the present invention and a pharmaceutically acceptable carrier, excipient or adjuvant.

[0027] 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 the pharmaceutical composition of the present invention.

[0028] According to another aspect, there is provided a kit comprising at least one reagent of the present invention.

[0029] According to some embodiments, the reagent is selected from antigen-binding fragments of antibodies, Fab fragments, single-chain antibodies, single-domain antibodies, small molecules, and peptides that specifically bind to CD28.

[0030] According to some embodiments, the reagent is less than 50 kDa.

[0031] According to some embodiments, the single-domain antibody is a camelid antibody or a shark antibody.

[0032] According to some embodiments, the camelid antibody comprises three CDRs, wherein:

[0033] 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);

[0034] 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

[0035] 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).

[0036] According to some embodiments, the camelid antibody comprises a sequence selected from:

[0037] a. EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVTVSS (SEQ ID NO:30);

[0038] b. EVQLVESGGGLVQAGGSLRLSCAASGSLFSINAMAWYRQAPGKQRELVAAITSSGSTNYANSVKGRFTVSRDNAKNTMYLQMNSLKPEDTAVYYCVVDEYGSDYWIWGQGTQVTVSS (SEQ ID NO:31); and

[0039] c. QVQLVESGGGLVQAGGSLRLSCAASGSIFSINAMGWYRQAPGKQRERVAAITSGGSTNYADSVKGRFTISRDNAKNTVYLQMNNLEPRDAGVYYCVVDLYGEDYWIWGQGTQVTVSS (SEQ ID NO:32).

[0040] According to some embodiments, the reagent comprises three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein:

[0041] 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).

[0042] According to some embodiments, the reagent is humanized.

[0043] According to some embodiments, the reagent is not a CD28 agonist.

[0044] According to some embodiments, the reagent is not a CD28 antagonist.

[0045] According to some embodiments, the reagent neither degrades mCD28 nor inhibits mCD28-mediated immune cell activation.

[0046] According to some embodiments, the antigen-binding fragment of the antibody does not induce antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0047] According to some embodiments, the reagent binds within the stalk region of CD28.

[0048] According to some embodiments, the stalk region comprises the amino acid sequence GKHLCPSPLFPGPSKP (SEQ ID NO: 9) or KGKHLCPSPLFPGPS (SEQ ID NO: 27).

[0049] According to some embodiments, the stalk region consists of the amino acid sequence HVKGKHLCPSPLFPGPSKP (SEQ ID NO: 10).

[0050] According to some embodiments, the reagent binds at least one protease at a cleavage site.

[0051] According to some embodiments, the reagent inhibits protease cleavage by at least one protease.

[0052] According to some embodiments, at least one protease is at least one metalloprotease.

[0053] According to some embodiments, at least one metalloprotease is MMP-2, MMP-13, or a combination thereof.

[0054] According to some embodiments, the subject has cancer.

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

[0056] According to some embodiments, the cancer is selected from melanoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, and colorectal cancer.

[0057] According to some embodiments, the method does not reduce mCD28 or reduces mCD28-mediated immune cell activation.

[0058] According to some embodiments, the subject's blood before administration comprises at least 5 ng / ml sCD28.

[0059] According to some embodiments, obtaining is obtaining a reagent that is less than 50 kDa, and wherein the obtained reagent is less than 50 kDa.

[0060] According to some embodiments, the method further comprises testing the ability of the reagent to block proteolytic cleavage of mCD8 on the cell surface.

[0061] According to some embodiments, the protease is selected from MMP-2 and MMP-13.

[0062] According to some embodiments, obtaining the reagent comprises at least one of the following steps:

[0063] a. Immunizing a shark or a camelid with the extracellular domain of CD28 or a fragment thereof, and collecting antibodies from the immunized organism; and

[0064] b. Screening a library of reagents that bind to the extracellular domain of CD28 or a fragment thereof and selecting the bound reagents.

[0065] According to some embodiments, the extracellular domain of CD28 or a fragment thereof is dimeric or monomeric.

[0066] According to some embodiments,

[0067] a. Collecting antibodies comprises extracting B cells from the spleen of the immunized shark or camelid; or

[0068] b. Selecting the bound reagents comprises sequencing the selected reagents and generating a recombinant form of the reagent from the sequence.

[0069] According to some embodiments, the method further comprises determining mCD28 downstream signaling in the presence of the obtained reagent and selecting at least one reagent that neither substantially agonizes nor substantially antagonizes mCD28 signaling.

[0070] According to some embodiments, the kit further comprises at least one of the following:

[0071] a. anti-PD-1 and / or PD-L1 immunotherapy; and

[0072] b. a label that describes the use of the reagent of the present invention in PD-1 and / or PD-L1 based immunotherapy.

[0073] From the detailed description given below, other embodiments and the full scope of applicability of the present invention will become apparent. However, it should be understood 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 be apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 . Soluble CD28 is produced during PBMC stimulation and is counteracted by adding protease inhibitors (PI). . The bar graph of the amount of soluble CD28 in cultures of PBMCs stimulated with SEB (0.5 ng / mL, left) or CMV peptide (0.5 μg / mL, right) was quantified by human CD28 ELISA (upper panel). A mixture of protease inhibitors was added at the indicated concentrations. Overall health and effector activity were examined by the secretion of interferon γ (lower panel).

[0075] Figure 2 . Soluble CD28 is produced during T cell stimulation by PHA and is counteracted by adding protease inhibitors. counteracted . Bar graphs of Jurkat cells (upper left) or isolated human CD4 T cells (upper right) stimulated with increasing concentrations of PHA (1 - 4 μg / mL, upper panel) in the presence of a protease inhibitor mixture at a fixed concentration (2 μM). In another setting, a fixed concentration of PHA was used to stimulate Jurkat T cells (1 μg / mL PHA, lower left) or human CD4 T cells (2 μg / mL PHA, lower right), and the concentration of the protease inhibitor mixture was titrated (0.5 - 2 μM). The concentration of human CD28 in the supernatant was quantified by standard sandwich ELISA.

[0076] Figure 3A - 3B . During the activation of human PBMC by SEB, specific ADAM - 10 and ADAM - 17 inhibitors abolish the accumulation of soluble CD28 without compromising their viability. During the activation of human PBMC by SEB, specific ADAM - 10 and ADAM - 17 inhibitors abolish the accumulation of soluble CD28 without compromising their viability. . Bar graphs of human PMBCs stimulated with SEB (1 ng / mL) in the presence of (3A - B) an ADAM - 10 - specific inhibitor (GI254023X) and (3B) an ADAM - 17 - specific inhibitor (TMI - 1) at various concentrations (0.01 - 1 μM). Cell viability in different treatments was evaluated using the MTT assay (upper panel). The concentration of human CD28 in the supernatant was quantified using a standard sandwich ELISA (lower panel).

[0077] Figure 4A - 4D . Soluble CD28 is produced during PBMC stimulation. (4A) Bar graphs of immature dendritic cells mixed with CD3 T cells from the same donor at a ratio of 1:5, without CMV peptides (black bars) or with CMV peptides (dark gray bars). Controls for each cell population alone or with CMV are light gray bars. The concentration of human CD28 in the supernatant was quantified using a standard sandwich ELISA. (4B - D) Bar graphs of human PBMCs stimulated with (4B) CMV or (4C) SEB or (4D) SEB in the presence of ADAM - 10 and ADAM - 17 inhibitors for 24 h and then transferred to a clean culture. Figure 4D The measurements in were at 120 h after cell transfer.

[0078] Figure 5 . Soluble CD28 inhibits effector cytokine secretion. . Bar graphs of human PBMCs stimulated with CMV (0.5 μg / mL), without recombinant human CD28 (black bars) or with recombinant human CD28 at the indicated concentrations (gray bars). Samples used for the experiment for the first time without CMV stimulation are indicated by light gray bars. The concentration of human IFNγ in the supernatant was quantified using a standard sandwich ELISA (Biolegend).

[0079] Figure 6 . Soluble CD28 increases IL - 6 cytokine secretion. . Bar graphs of human PBMCs stimulated with CMV (0.5 μg / mL), without recombinant human soluble CD28 (black bars) or with recombinant human soluble CD28 at the indicated concentrations (gray bars). Samples used for the experiment for the first time without CMV stimulation are indicated by light gray bars. The concentration of human IL - 6 in the supernatant was quantified using a standard sandwich ELISA (Biolegend).

[0080] Figure 7A - 7E.(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. Concentrations of human IL-6, IFNγ, and IL-4 in the supernatant were quantified using a standard sandwich ELISA (Biolegend). Concentrations of human IL-8, IL-12p(40), and IL-10 in the supernatant were quantified using a multiplex assay with a Magpix system (Millipore). (7B) Bar graph of cytokine secretion of autologous monocytes and CD3 MLR. Samples used for the experiment for the first time without CMV stimulation are indicated by light gray bars. CMV alone or with IgG control is indicated by black bars. Increasing concentrations of sCD28 are indicated by dark gray bars. (7C) Line graph of lymphocyte cluster formation of human PBMCs stimulated with SEB or with control IgG (gray triangles) in the presence of recombinant human soluble CD28 (gray circles). (7D) Bar graph of IDO secreted into the culture measured from monocytes treated with and without recombinant human sCD28 using a kynurenine ELISA kit. (7E) Scatter plot of intracellular FACS of IDO in monocytes treated with and without recombinant human sCD28.

[0081] Figure 8A - 8C . Soluble CD28 hampers anti - PD1 treatment. . (8A) Bar graph of human PBMCs stimulated with SEB (200 ng / mL, left bars) or CMV peptide (0.5 μg / mL, right bars) for 3 days in the presence of anti-PD1 (MK3475, 5 μg / mL, black bars) or recombinant human soluble CD28 (2 and 10 μg / mL, gray bars) or a combination of both (dashed bars). (8B) Bar graph of cytokine secretion of a monocyte MLR setup, samples used for the experiment for the first time - white bars, CMV alone - light gray bars, sCD28 - black bars, MK-3475 - dark gray bars, sCD28 + MK-3475 - checkered bars. Concentrations of human IFNγ, TGFβ, and IL-2 in the supernatant were quantified using a standard sandwich ELISA (Biolegend). (8C) Histograms of surface PD-L1 (left) and PD-L2 (right) expression in monocytes after incubation with control and sCD28.

[0082] Figure 9A - 9C . Soluble CD28 in cancer patients . (9A) Dot plots showing 10 cancer indications and 20 plasma samples in each of healthy donors for investigating the presence of soluble human CD28. Samples containing high levels of soluble CD28 were re-examined with multiple dilution factors. The concentration of human CD28 in the supernatant was quantified using a standardized sandwich ELISA, internally calibrated to accommodate readings of human plasma samples. (9B) Bar graphs of IFNγ secretion from SEB-stimulated PBMCs from cancer patients measured by sandwich ELISA in the presence of sCD28, MK-3475, and a combination of both (sarcoma patients - upper left, renal cancer patients - upper right, and two different head and neck cancer patients - lower). (9C) Bar graphs of viability and proliferation of cancer cells SCC-25 alone, with IL-6, co-cultured with monocytes, or co-cultured with monocytes and sCD28.

[0083] Figure 10A - 10B . (10A) Bar graphs of IFNγ from isolated CD3 T cells stimulated with anti-CD3 in the presence of a constant level of CD80-Fc and titrated soluble CD28. (10B) Isolated PBMCs stimulated with CMV in the presence of a constant level of sCD28 and titrated CD80-Fc.

[0084] Figure 11A - 11B . (11A - B) Line graphs of tumor volume of H22 cells inoculated in immunocompetent mice treated with anti-PD-1 antibody, without administration of recombinant mouse CD28 (11A) and with administration of recombinant mouse CD28 (11B).

[0085] Figure 12A - 12C.(12A) A line graph showing the antigen binding of the CD28 stem region dimer peptide conjugated to M9 with BSA (right) and recombinant human CD28 protein (left) by serial dilution cloning. The antigen was immobilized on a maxisorp ELISA plate. A dilution series of clone M9 was performed, and the bound antibody was detected using donkey anti-mouse IgG (H&L)-HRP and developed using TMB. (12B) A bar graph of ELISA detection of recombinant human sCD28 (left) and sCD28 shed from human PBMC activated with SEB (right). ELISA used antibody #3 as a positive control (2 μg / mL, grey bar), irrelevant antibody M39 as a negative control (10 μg / mL, dark grey bar), and anti-cleaved antibody M9 (10 μg / mL, black bar). Detection of recombinant CD28 or shed CD28 was performed by using an ELISA kit conjugated to HRP (0.5 μg / mL) to detect the antibody. (12C) A histogram showing the binding of antibody M9 (top) and control antibody CD28.2 (bottom) at a fixed concentration of 10 μg / ml (black histogram) to human CD28 expressed in mouse HEK293 cells. Polyclonal mouse IgG was used as a negative control (10 μg / ml) and depicted in the grey histogram. Detection was completed by secondary incubation with Alexa Fluor 647-conjugated goat anti-mouse.

[0086] Figure 13 . Binding to the human CD28 stem region sequence by ELISA. Antigen binding was analyzed by serial dilution of different VHH clones. The biotin-conjugated CD28 stem region dimer peptide used as the antigen was immobilized on a neutravidin-coated ELISA maxi-sorb plate. A dilution series of VHH clones was performed, and the bound VHH was detected using an anti-His tag-HRP conjugated antibody and developed using TMB.

[0087] Figure 14 . Binding of VHH#2A1 to membrane human CD28. HEK cells overexpressing human CD28 were incubated with FITC-conjugated VHH clone 2A1 (50 μg / mL, black histogram) and FITC-conjugated isotype control (mIgG, 50 μg / mL, grey histogram). Binding was evaluated by FACS analysis.

[0088] Figure 15. Anti-CD28 stem region VHH clones do not block ligand binding to membrane CD28. HEK293 cells overexpressing human CD28 were monitored by flow cytometry for CD86-Fc (2 μg / mL) binding using a secondary anti-human Fc antibody conjugated to AlexaFlour 647. Addition of anti-CD28 VHH clones (30 μg / mL, black histogram) to CD86-Fc did not alter the magnitude of CD86 binding, while addition of the commercial antibody clone CD28.2 (10 μg / mL, upper left panel, black histogram) significantly reduced binding.

[0089] Figure 16 . Assessment of the agonist effect of anti-CD28 VHH clones. Human isolated CD3 cells were stimulated with plate-bound anti-CD3 (OKT3, 2 μg / mL, light gray bars) for 2 days in the presence of the anti-CD28 agonist antibody clone 28.2 (2 μg / mL, dark gray) used as a positive control, anti-CD28 stem region VHH, or an irrelevant VHH clone (20 μg / mL, black bars). The concentration of human IFNγ secreted into the supernatant was quantified using a standard sandwich ELISA (Biolegend).

[0090] Figure 17 . In vitro blockade of MMP-2-mediated cleavage of the human CD28 stem region by VHH clones. c-Myc-conjugated and biotinylated human CD28 stem region dimer peptide (1 μM) was incubated with 50 ng rhMMP-2 for 5 hours in the presence of the MMP-2 inhibitor (TMI-1, 50 nM) M9 Fab or indicated VHH clones at various concentrations (0.4 - 10 μg / mL). The mixture was loaded onto a neutravidin-coated ELISA maxi-sorb plate, followed by extensive washing and detection of the intact peptide with an anti-cMyc-HRP-conjugated antibody and developed with TMB.

[0091] Figure 18 . Anti-CD28 stem region VHH clones 2A1 and 4A4 inhibit CD28 shedding in HEK cells overexpressing human CD28. The level of soluble CD28 was measured in the medium of HEK cells stably expressing human CD28 after 48-hour incubation. The effect of different treatments with the MMP inhibitor (TMI-1, 1 μM, dark gray bars), negative control with an irrelevant VHH (upper left panel, black bars), or anti-CD28 stem region VHH clones (black bars) at various concentrations (3.3 - 100 μg / mL) on the amount of soluble CD28 was depicted. The level of soluble human CD28 in the supernatant was quantified using a standard sandwich ELISA (R&D Systems).

[0092] Figure 19. Anti-CD28 stem region VHH clones 2A1 and 4A4 inhibit CD28 shedding in isolated CD4 T cells activated by PHA and IL2. The levels of soluble CD28 were measured in the medium of isolated human CD4 T cells stimulated with 5 μg / mL PHA and 200 IU / mL IL-2 (light grey bars). The effects of different treatments with various concentrations (0.4 - 50 μg / mL) of an MMP inhibitor (TMI-1, 1 μM, dark grey bars), a negative control of an irrelevant VHH (upper left panel, black bars), anti-CD28 stem region VHH clones or the Fab format of antibody M9 clone (black bars) on the amount of soluble CD28 were depicted. The levels of soluble human CD28 in the supernatant were quantified using a standard sandwich ELISA (R&D Systems).

[0093] Figure 20 . Anti-CD28 stem region VHH clones 2A1 and 4A4 inhibit CD28 shedding in PBMCs activated by superantigen. The levels of soluble CD28 were measured in the medium of isolated PBMCs stimulated with 1 ng / mL SEB (light grey bars). The effects of different treatments with various concentrations (0.4 - 50 μg / mL) of an MMP inhibitor (TMI-1, 1 μM, dark grey bars), a negative control of an irrelevant VHH (upper left panel, black bars), anti-CD28 stem region VHH clones or the M9 clone in Fab format (black bars) on the amount of soluble CD28 were depicted. The levels of soluble human CD28 in the supernatant were quantified using a standard sandwich ELISA (R&D Systems).

[0094] Figure 21 . Assessment of the antagonist effect of anti-CD28 VHH clones. Human isolated CD3 cells were stimulated with plate-bound anti-CD3 (OKT3, 2 μg / mL, light grey bars) for 24 hours in the presence of recombinant CD80-Fc protein (5 μg / mL, dark grey bars) used as a ligand for CD28 co-stimulation. An irrelevant VHH clone (upper left panel) or anti-CD28 stem region VHH was added at various concentrations (3.75 - 30 μg / mL, black bars). The concentration of human IL-2 in the supernatant was quantified using a standard sandwich ELISA (Biolegend).

[0095] Figure 22. In vitro blocking activity of VHH clone 2A1 against human CD28 stem region cleavage by MMP-13. c-Myc and biotinylated human CD28 stem region dimer peptide (1 μM) were incubated with 50 ng rhMMP-13 (light gray bars) in the presence of MMPi (TMI-1, 50 nM, dark gray bars), an irrelevant VHH clone (black bars in the left panel), or VHH clone 2A1 (black bars in the right panel) at various concentrations (0.62 - 10 μg / mL) for 5 hours. The mixtures were loaded onto a neutravidin-coated ELISA maxi-sorb plate, followed by extensive washing and detection of intact peptide with an anti-cMyc-HRP conjugated antibody, and developed with TMB.

[0096] Figure 23 . Anti-CD28 stem 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 the WT sequence of the human CD28 stem region or to the L145-mutated sequence by direct ELISA. Biotin-conjugated wild-type or L145K CD28 stem region dimer peptides were immobilized on a neutravidin-coated ELISA maxi-sorb plate. Serial dilutions of VHH clones (0.2 - 5 μg / mL) and an irrelevant VHH clone (upper left panel) were performed, and bound VHH was detected with an anti-His tag-HRP conjugated antibody and developed with TMB. Detailed Description

[0097] In some embodiments, the present invention provides a reagent less than 100 kilodaltons (kDa) that binds to membrane CD28 (mCD28) on the cell surface and inhibits protease cleavage of mCD28. Also provided are methods of treating cancer, improving PD-1 / PD-L1-based immunotherapy, and reducing sCD28 levels in a subject, including administering the reagent of the present invention. The reagents and methods of the present invention are based on the surprising finding that full-size antibodies against the cleavage site of mCD28 are too large to access the membrane-proximal region and thus cannot inhibit shedding. Instead, smaller reagents specific for mCD28 on the cell surface are needed. Further, a large number of cancer patients have elevated sCD28 levels in their bloodstream, which is caused by sCD28 shedding. This sCD28 acts as an immunosuppressant, and thus reduction of shedding has the dual benefit of reducing inhibition by sCD28 and increasing immune activation through mCD28 signaling. Further, it was unexpectedly found that sCD28 can inhibit PD-1 / PD-L1-based immunotherapy.

[0098] Reagents

[0099] According to a first aspect, there is provided a reagent that binds to membrane CD28 (mCD28) and inhibits proteolytic cleavage of mCD28.

[0100] In some embodiments, mCD28 is on the cell surface. In some embodiments, mCD28 is in the membrane. In some embodiments, the reagent is not a full-size antibody. In some embodiments, the reagent is not IgG. In some embodiments, the reagent is less than 100 kilodaltons (kDa). In some embodiments, the reagent 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 reagent is less than 50 kDa. In some embodiments, the reagent is less than 25 kDa. In some embodiments, the reagent is less than 20 kDa. In some embodiments, the reagent is less than 15 kDa.

[0101] In some embodiments, CD28 is mammalian CD28. In some embodiments, CD28 is human CD28. In some embodiments, human CD28 comprises or consists of the following amino acid sequence: MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:1). In some embodiments, mature CD28 lacks the signal peptide and comprises the sequence: NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:2).

[0102] In some embodiments, the DNA coding sequence encoding full-length human CD28 comprises the sequence: ATGCTCAGGCTGCTCTTGGCTCTCAACTTATTCCCTTCAATTCAAGTAACAGGAAACAAGATTTTGGTGAAGCAGTCGCCCATGCTTGTAGCGTACGACAATGCGGTCAACCTTAGCTGCAAGTATTCCTACAATCTCTTCTCAAGGGAGTTCCGGGCATCCCTTCACAAAGGACTGGATAGTGCTGTGGAAGTCTGTGTTGTATATGGGAATTACTCCCAGCAGCTTCAGGTTTACTCAAAAACGGGGTTCAACTGTGATGGGAAATTGGGCAATGAATCAGTGACATTCTACCTCCAGAATTTGTATGTTAACCAAACAGATATTTACTTCTGCAAAATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCTGA (SEQ ID NO:3).

[0103] As used herein, sCD28 refers to any CD28 fragment or variant that does not include a transmembrane domain and thus cannot integrate in the membrane. In some embodiments, the CD28 transmembrane domain includes the amino acid sequence FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:4). In some embodiments, sCD28 is not membrane-bound. In some embodiments, sCD28 is in solution. In some embodiments, sCD28 is CD28 in the blood. In some embodiments, sCD28 is CD28 in the TME. In some embodiments, sCD28 is CD28 in the body fluid. In some embodiments, sCD28 lacks exon 3 of CD28. In some embodiments, sCD28 is a splice variant generated by alternative splicing that splices out 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 generated by alternative splicing of CD28. In some embodiments, alternative splicing splices out exon 3 of CD28. In some embodiments, sCD28 includes the amino acid sequence: MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGEE (SEQ ID NO:5). In some embodiments, sCD28 consists of the amino acid sequence of SEQ ID NO:5. In some embodiments, sCD28 lacks a signal peptide and includes the sequence: NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGEE (SEQ ID NO:6). In some embodiments, sCD28 consists of the amino acid sequence of SEQ ID NO:6.In some embodiments, sCD28 comprises the amino acid sequence: MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSP (SEQ ID NO:48). In some embodiments, sCD28 consists of the amino acid sequence of SEQ ID NO:48. In some embodiments, sCD28 lacks a signal peptide and comprises the sequence: NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSP (SEQ ID NO:49). In some embodiments, sCD28 consists of the amino acid sequence of SEQ ID NO:49.

[0104] In some embodiments, the DNA coding sequence encoding human sCD28 comprises the sequence: ATGCTCAGGCTGCTCTTGGCTCTCAACTTATTCCCTTCAATTCAAGTAACAGGAAACAAGATTTTGGTGAAGCAGTCGCCCATGCTTGTAGCGTACGACAATGCGGTCAACCTTAGCTGCAAGTATTCCTACAATCTCTTCTCAAGGGAGTTCCGGGCATCCCTTCACAAAGGACTGGATAGTGCTGTGGAAGTCTGTGTTGTATATGGGAATTACTCCCAGCAGCTTCAGGTTTACTCAAAAACGGGGTTCAACTGTGATGGGAAATTGGGCAATGAATCAGTGACATTCTACCTCCAGAATTTGTATGTTAACCAAACAGATATTTACTTCTGCAAAATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCTGA (SEQ ID NO:7).

[0105] The effects of sCD28 on immune cells are well known in the art and include, as non-limiting examples, induction of immune cells of anti-inflammatory cytokines such as IL-10 or TGFβ, expression of immune cells of indoleamine 2,3-dioxygenase (IDO), and downregulation of immune cells of pro-inflammatory cytokines such as IL-2 or IFN-γ. In some embodiments, inhibiting proteolytic cleavage of membrane CD28 by the reagent includes inhibiting the production of sCD28. In some embodiments, inhibiting the production of sCD28 includes inhibiting the effects of sCD28 on immune cells.

[0106] As used herein, inhibiting protease cleavage refers to any reduction in the protease cleavage of mCD28. In some embodiments, the inhibition is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% reduction in cleavage. Each possibility represents a separate embodiment of the invention. In some embodiments, inhibiting protease cleavage maintains the level of mCD28 on immune cells. In some embodiments, inhibiting protease cleavage increases the level of mCD28 on immune cells. In some embodiments, inhibiting protease cleavage maintains an mCD28 level sufficient for immune stimulation.

[0107] In some embodiments, the reduction in protease cleavage is a reduction in the cleavage of at least one protease. In some embodiments, the reduction in protease cleavage is a reduction in the cleavage of 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.

[0108] In some embodiments, the reagent is selected from 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 reagent is a Fab fragment. In some embodiments, the reagent is a single-chain antibody. In some embodiments, the reagent is a single-domain antibody. In some embodiments, the reagent is a peptide that specifically binds to CD28.

[0109] In some embodiments, the reagent lacks an Fc domain. In some embodiments, the reagent is an antigen-binding domain that lacks an Fc domain. In some embodiments, the reagent is a camelid antibody, shark antibody or nanobody. In some embodiments, the antibody or fragment is fused to another protein or a fragment of a protein. In some embodiments, the second protein or fragment increases the half-life, particularly in serum. In some embodiments, the half-life extending protein is human serum albumin. In some embodiments, the reagent is modified by generating a modified chemical that enhances the half-life. In some embodiments, the modification is PEGylation and the chemical is polyethylene glycol. Those skilled in the art will recognize that any half-life extending protein or chemical agent, or modification known in the art, can be used.

[0110] Examples of the reagent 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 reagent 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 reagent 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 reagent is a single-domain antibody. In some embodiments, the reagent is a nanobody. In some embodiments, the reagent is a VHH antibody. As used herein, the terms "single-domain antibody", "nanobody", and "VHH antibody" are synonyms and are used interchangeably. In some embodiments, the peptide has specificity for binding to CD28. In some embodiments, the reagent is a peptide that specifically binds to CD28. In some embodiments, the peptide is selected from antibodies, antigen-binding fragments of antibodies, Fab fragments, single-chain antibodies, single-domain antibodies, nanobodies, VHH antibodies, and antibody mimetics. As used herein, the term "antibody mimetic" refers to an organic compound that can specifically bind to a target antigen. In some embodiments, the antibody mimetic is not structurally related to an antibody. Examples of antibody mimetics include, but are not limited to, affilins, affimers, affitins, alphabodies, anticalins, avimers, DARPins, fynomers, Kunitz domain peptides, monobodies, and nanoclamps. In some embodiments, the antibody mimetic is a DARPin. All of these reagents are known in the art and are known to be used to block the interaction between a receptor and its ligand. Small molecules and proteins that can bind to mCD28 can occlude the cleavage site or may cause steric hindrance or impair protease access. In some embodiments, the protein is an antibody mimetic. As used herein, the term "DARPin" refers to designed ankyrin repeat proteins. DARPins are generally engineered antibody mimetic proteins that generally have a high degree of specificity for their protein target. Thus, a DARPin of CD28 can be an example of a reagent.

[0111] In some embodiments, the Fab fragment has a size of about 50 kDa. In some embodiments, the Fab fragment has a size less than 100 kDa. In some embodiments, the Fab fragment has a size less than 80 kDa. In some embodiments, the Fab fragment has a size less than 70 kDa. In some embodiments, the Fab fragment has a size less than 50 kDa. In some embodiments, the Fab fragment has a size of 50 kDa or less. In some embodiments, the single-chain antibody has a size of about 25 kDa. In some embodiments, the single-chain antibody has a size less than 50 kDa. In some embodiments, the single-chain antibody has a size less than 40 kDa. In some embodiments, the single-chain antibody has a size less than 30 kDa. In some embodiments, the single-chain antibody has a size less than 25 kDa. In some embodiments, the single-chain antibody has a size of 25 kDa or less. In some embodiments, the single-domain antibody has a size of about 15 kDa. In some embodiments, the single-domain antibody has a size between 10 - 17 kDa. In some embodiments, the single-domain antibody has a size between 10 - 16 kDa. In some embodiments, the single-domain antibody has a size between 10 - 15 kDa. In some embodiments, the single-domain antibody has a size between 12 - 15 kDa. In some embodiments, the single-domain antibody has a size between 12 - 16 kDa. In some embodiments, the single-domain antibody has a size between 12 - 17 kDa. In some embodiments, the single-domain antibody has a size less than 25 kDa. In some embodiments, the single-domain antibody has a size less than 20 kDa. In some embodiments, the single-domain antibody has a size less than 15 kDa. In some embodiments, the single-domain antibody has a size of 15 kDa or less. Due to its small size and only having 3 CDRs, the single-domain antibody has a convex shape and binds its epitope from only one side. In contrast, the Fab fragment and the single-chain antibody contain 6 CDRs and bind the epitope from at least two sides. In some embodiments, binding only with 3 CDRs allows better access to the mCD28 stem region compared to binding with 6 CDRs. In some embodiments, the geometry of the binding of the single-domain antibody is more favorable for accessing the mCD28 stem region.

[0112] As used herein, the term "antibody" refers to a polypeptide or a group of polypeptides that includes at least one binding domain formed by the folding of a polypeptide chain having a three-dimensional binding space with an inner surface shape and charge distribution complementary to the characteristics of an epitope. Antibodies typically have a tetrameric form, comprising two pairs of identical polypeptide chains, each pair having one "light" chain and one "heavy" chain. The variable regions of each light / heavy chain pair form the antibody binding site. Antibodies can be oligoclonal, polyclonal, monoclonal, chimeric, camelized, CDR-grafted, multispecific, bispecific, catalytic, humanized, fully human, anti-idiotypic and antibodies that can be labeled in soluble or bound form, as well as fragments (including epitope-binding fragments), variants or derivatives thereof, alone or in combination with other amino acid sequences. Antibodies can be from any species. The term antibody also includes binding fragments, including, but not limited to, Fv, Fab, Fab', F(ab')2, single-chain antibody (scFv), diabody and disulfide-stabilized Fv (dsFv). Specifically, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing an antigen-binding site. Antibody fragments can be fused or not fused to another immunoglobulin domain, including, but not limited to, the Fc region or fragments thereof. Those skilled in the art will further recognize 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.

[0113] Immunoglobulin molecules can have any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0114] The basic unit of a naturally occurring antibody structure is a heterotetrameric glycoprotein complex of approximately 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains, associated non-covalently and linked together by disulfide bonds. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. There are five human antibody classes (IgG, IgA, IgM, IgD, and IgE), and within these classes, various subclasses are identified based on structural differences, such as the number of immunoglobulin units in a single antibody molecule, the disulfide bond structure of the individual units, and 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 invention. In some embodiments, the Fab fragment has a size of less than 50 kDa.

[0115] The amino-terminal regions of the heavy and light chains are more diverse in sequence than the carboxyl-terminal regions and are thus called variable domains. This part of the antibody structure confers the antigen-binding specificity of the antibody. The heavy variable (VH) domain and the light variable (VL) domain together form a single antigen-binding site; thus, the basic immunoglobulin unit has two antigen-binding sites. Specific amino acid residues are thought to form an interface between the light and heavy chain variable domains (Chothia et al., J. Mol. Biol. 186, 651-63 (1985); Novotny and Haber, (1985) Proc. Natl. Acad. Sci. USA 82, 4592-4596).

[0116] The carboxyl-terminal portions of the heavy and light chains form constant domains, namely CH1, CH2, CH3, CL. Although there is less diversity in these domains, there are differences between animal species and further, within the same individual, there are several different isotypes of antibodies, each with different functions.

[0117] The term "framework region" or "FR" refers to the amino acid residues in the variable domain of an antibody that are different from the amino acid residues of the hypervariable regions as defined herein. The term "hypervariable region" as used herein refers to the amino acid residues in the variable domain of an antibody that are responsible for antigen binding. The hypervariable regions include the amino acid residues from "complementary determining regions" or "CDRs". The CDRs are mainly responsible for binding to the epitope of the antigen. The boundaries of the FRs and CDRs have been precisely defined (see, Kabat et al.).

[0118] Immunoglobulin variable domains can also be analyzed using the IMGT information system (www: / / imgt.cines.fr / )( / V-Quest) to identify variable segments, including CDRs. See, e.g., Brochet, X. et al, Nucl. Acids Res. 36:W503-508 (2008).

[0119] Chothia et al. also defined a numbering system applicable to the variable domain sequences of any antibody. A person skilled in the art can unambiguously assign such a "Chothia numbering" system to any variable domain sequence without relying on any experimental data other than the sequence itself. As used herein, "Chothia numbering" refers to the numbering system proposed by Chothia et al. in the Journal of Molecular Biology, "Canonical Structures for the Hypervariable regions of immunoglobulins" (1987) and Chothia in Nature, "Conformations of Immunoglobulin Hypervariable Regions" (1989).

[0120] As used herein, the terms "single-chain antibody" and "single-chain variable fragment" are used synonymously and refer to a fusion protein of the variable regions of the heavy and light chains of an immunoglobulin 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 between 10 and 25 amino acids. In some embodiments, the linker is between 1-40, 5-40, 10-40, 1-35, 5-35, 10-35, 1-30, 5-30, 10-30, 1-25, 5-25, or 10-25 amino acids. Each possibility represents a separate embodiment of the present invention. In some embodiments, the single-chain antibody 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 CDR of antibody M9.

[0121] As used herein, the terms "single-domain antibody", "nanobody", and "VHH" are used synonymously and refer to an antibody fragment composed of a single monomer variable antibody domain. In some embodiments, the single-domain antibody is a camelid antibody. In some embodiments, the camelid is a camel, llama, or alpaca. In some embodiments, the camelid is a camel. In some embodiments, the camelid is a llama. In some embodiments, the camelid is an alpaca. In some embodiments, the single-domain antibody is a shark antibody.

[0122] Furthermore, as indicated herein, the amino acid residues of the nanobody are numbered according to the general VH numbering given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, Md., Publication No. 91), which has been applied to the VHH domain of camelid antibodies in the articles of Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun. 23; 240(1-2): 185-195; or as mentioned herein. According to this numbering, the FR1 of the nanobody includes the amino acid residues at positions 1-30, the CDR1 of the nanobody includes the amino acid residues at positions 31-35, the FR2 of the nanobody includes the amino acids at positions 36-49, the CDR2 of the nanobody includes the amino acid residues at positions 50-65, the FR3 of the nanobody includes the amino acid residues at positions 66-94, the CDR3 of the nanobody includes the amino acid residues at positions 95-102, and the FR4 of the nanobody includes the amino acid residues at positions 103-113. In this regard, it should be noted that - as is well known in the art for VH domains and VHH domains - the total number of amino acid residues in each CDR may 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, generally, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence. Generally, however, it can be said that, according to the Kabat numbering, and regardless of the number of amino acid residues in the CDRs, 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.

[0123] An alternative method of numbering the amino acid residues of the VH domain (which can also be applied in a similar manner to the VHH domain of camelid antibodies and nanobodies) is the method described by Chothia et al. (Nature 342, 877-883 (1989)), the so-called "AbM definition" and the so-called "contact definition". However, in the present specification, aspects and figures, unless otherwise stated, the Kabat-based numbering used by Riechmann and Muyldermans for the VHH domain will be followed.

[0124] As used herein, the term "humanized antibody" refers to an antibody from a non-human species whose protein sequence has been modified to increase its similarity to a human antibody. A humanized antibody can be produced by generating recombinant DNA encoding the CDRs of a non-human antibody surrounded by sequences of a human-like antibody. In some embodiments, a humanized antibody is a chimeric antibody. In some embodiments, humanization includes inserting the CDRs of the present invention into a human antibody scaffold or framework. Humanized antibodies are well known in the art and any method for producing a humanized antibody that retains the CDRs of the present invention can be used.

[0125] The term "monoclonal antibody" or "mAb" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for variants that may arise during the production of the monoclonal antibody, which are typically present in minor amounts. In contrast to polyclonal antibody preparations, which generally include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on an antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody obtained from a substantially homogeneous population of antibodies and should not be construed as indicating any particular method of preparation. Monoclonal antibodies used in accordance with the methods provided herein can be prepared by the hybridoma method first described by Kohler et al. (Nature 256:495 (1975)), or can be prepared by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). For example, "monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described by Clackson et al. (Nature 352:624-628 (1991)) and Marks et al. (J. Mol. Biol. 222:581-597 (1991)).

[0126] The mAbs of the present invention can be of any immunoglobulin class, including IgG, IgM, IgD, IgE, or IgA. Hybridomas producing mAbs can be cultured in vitro or in vivo. High titers of mAbs can be obtained in in vivo production, where cells from a single hybridoma are injected intraperitoneally into pristane-primed Balb / c mice to produce ascites containing high concentrations of the desired mAb. mAbs of isotype IgM or IgG can be purified from these ascites fluids or culture supernatants using column chromatography well known to those skilled in the art.

[0127] "Antibody fragment" includes a portion of a full antibody, preferably including its antigen-binding region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; tandem diabodies (taDb); linear antibodies (e.g., U.S. Patent No. 5,641,870, Example 2; Zapata et al. Protein Eng. 8(10):1057-1062 (1995)); single-arm antibodies, single variable domain antibodies, minibodies, single-chain antibody molecules; multispecific antibodies formed from antibody fragments (e.g., including but not limited to, Db-Fc, taDb-Fc, taDb-CH3, (scFv)4-Fc, di-scFv, bi-scFv, or tandem (di,tri)-scFv); and bispecific T cell engagers (BiTE).

[0128] Papain digestion of an antibody produces two identical antigen-binding fragments, called "Fab" fragments, each having a single antigen-binding site and a residual "Fc" fragment, the name of which reflects its ability to crystallize readily. Pepsin treatment yields F(ab')2 fragments, which have two antigen-binding sites and are still capable of cross-linking antigens.

[0129] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and antigen-binding site. This region consists of a dimer of one heavy-chain and one light-chain variable domain, which are tightly, non-covalently associated. The three surfaces of the VH-VL dimer are in this configuration. Collectively, six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv that contains only three hypervariable regions specific for the antigen) has the ability to recognize and bind the antigen, although with a lower affinity than the entire binding site.

[0130] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CHl) of the heavy chain. The Fab' fragment differs from the Fab fragment in that several residues are added at the carboxyl terminus of the heavy-chain CH1 domain, which includes one or more cysteines from the antibody hinge region. Fab'-SH is the name for Fab' herein, where the cysteine residue(s) of the constant domain carry at least one free thiol group. The F(ab')2 antibody fragment was originally produced as pairs of Fab' fragments with a hinge cysteine between them. Other chemical conjugations of antibody fragments are also known.

[0131] The "light chain" of an antibody (immunoglobulin) from any vertebrate species can be classified into one of two distinct types called κ and λ based on the amino acid sequence of its constant domain.

[0132] Antibodies can be assigned to different classes based on the amino acid sequence of the constant domains of their heavy chains. There are five main classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chains corresponding to the different classes of antibodies are designated α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0133] A "single-chain Fv" or "scFv" antibody fragment comprises 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 scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenberg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0134] The term "diabody" refers to a small antibody fragment having two antigen-binding sites, which fragment comprises a heavy-chain variable domain (VH) linked to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, these domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabody production is known in the art and is described in Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0135] The term "multispecific antibody" is used in the broadest sense and specifically encompasses antibodies having multi-epitope specificity. Such multispecific antibodies include, but are not limited to, antibodies comprising heavy-chain variable domains (VH) and light-chain variable domains (VL) - wherein the VHVL unit has multi-epitope specificity, antibodies having two or more VL and VH domains - wherein each VHVL unit binds to a different epitope, antibodies having two or more individual variable domains - wherein each individual variable domain binds to a different epitope, intact antibodies, antibody fragments (such as Fab, Fv, dsFv, scFv), diabodies, bispecific diabodies, triabodies, trifunctional antibodies, covalently or non-covalently linked antibody fragments. "Multi-epitope specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different target(s).

[0136] Monoclonal antibodies can be prepared using methods well known in the art. Examples include a variety of techniques such as those described in Kohler, G. and Milstein, C, Nature 256:495 - 497 (1975); Kozbor et al, Immunology Today 4:72 (1983); Cole et al, pg. 77 - 96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

[0137] In addition to the conventional methods of generating antibodies in vivo, phage display technology can be used to generate antibodies in vitro. Compared to conventional antibody production, the production of such recombinant antibodies is much faster and recombinant antibodies can be generated against a large number of antigens. Furthermore, when using conventional methods, many antigens have proven to be non - immunogenic or highly toxic and thus cannot be used to generate antibodies in animals. In addition, affinity maturation of recombinant antibodies (i.e., increasing affinity and specificity) is very straightforward and relatively rapid. Finally, a large number of different antibodies against a specific antigen can be generated in a single selection procedure. To generate recombinant monoclonal antibodies, one can use various display library - based methods to generate a large number of antibodies with different antigen - recognition sites. Such libraries can be constructed in a variety of ways: synthetic libraries can be generated by cloning synthetic CDR3 regions into heavy - chain germline gene libraries, thus generating large antibody libraries from which recombinant antibody fragments with various specificities can be selected. One can use libraries of human lymphocytes as starting materials for constructing antibody libraries. A naive library of human IgM antibodies can be constructed, thus creating a human library with a wide diversity. This method has been widely and successfully used to select a large number of antibodies against different antigens. Protocols for phage library construction and recombinant antibody selection are provided in the well - known reference text Current Protocols in Immunology, Colligan et al (Eds.), John Wiley & Sons, Inc. (1992 - 2000), Chapter 17, Section 17.1.

[0138] 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 human antibody or consensus antibody framework sequences. Further changes can then be introduced into the antibody framework to modulate affinity or immunogenicity.

[0139] In some embodiments, antibodies and parts thereof include: antibodies, antibody fragments, Fab and F(ab')2, single domain antigen binding recombinant fragments and natural nanobodies. In some embodiments, the antigen binding fragment is selected from Fv, Fab, F(ab')2, scFV or scFV2 fragments.

[0140] In some embodiments, the invention provides nucleic acid sequences encoding the antibodies or antigen-binding portions of the invention.

[0141] For example, a polynucleotide can encode a complete immunoglobulin molecule chain, such as a light chain or a heavy chain. A complete heavy chain includes not only a heavy chain variable region (VH) but also a heavy chain constant region (CH), which generally includes three constant domains: CH1, CH2 and CH3; and a "hinge" region. In some cases, the presence of a constant region is desirable.

[0142] Other polypeptides that can be encoded by polynucleotides include antigen-binding antibody fragments, such as single domain antibodies ("dAbs"), Fv, scFv, Fab' and CHI, and CK or CL domains have been removed. Because mini antibodies are smaller than conventional antibodies, all of them should achieve better tissue penetration in clinical / diagnostic use, but they should retain higher binding affinity than monovalent antibody fragments such as dAbs, which are bivalent. Therefore, unless the context otherwise provides, the term "antibody" used herein includes not only whole antibody molecules, but also antigen-binding antibody fragments of the types discussed above. Each framework region present in the encoded polypeptide may include at least one amino acid substitution relative to the corresponding human receptor framework. Therefore, for example, the framework region may include a total of three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions relative to the receptor framework region. In view of the characteristics of the individual amino acids comprising the disclosed protein product, those skilled in the art will recognize some reasonable substitutions. Amino acid substitutions, ie, "conservative substitutions," may be made, for example, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved.

[0143] Suitably, the polynucleotides described herein may be isolated and / or purified.In some embodiments, the polynucleotide is an isolated polynucleotide.

[0144] As used herein, the term "non-naturally occurring" substances, compositions, entities, and / or any combination of substances, compositions or entities, or any grammatical variations thereof, is a conditional term that expressly excludes, but only excludes, those forms of substances, compositions, entities, and / or combinations of substances, compositions, entities that are known to one of ordinary skill in the art as "naturally occurring" or that may at any time be determined or interpreted by a judge, administrative or judicial body to be "naturally occurring."

[0145] On the other hand, a reagent is provided that includes three CDRs, where CDR1 includes the amino acid sequence set forth in SEQ ID NO:33 (INAMG), CDR2 includes the amino acid sequence set forth in SEQ ID NO:34 (AISGGGDTYYADSVKG), and CDR3 includes the amino acid sequence set forth in SEQ ID NO:35 (DLYGSDYWD).

[0146] On the other hand, a reagent is provided that includes three CDRs, where CDR1 includes the amino acid sequence set forth in SEQ ID NO:36 (INAMA), CDR2 includes the amino acid sequence set forth in SEQ ID NO:37 (AITSSGSTNYANSVKG), and CDR3 includes the amino acid sequence set forth in SEQ ID NO:38 (DEYGSDYWI).

[0147] On the other hand, a reagent is provided that includes three CDRs, where CDR1 includes the amino acid sequence set forth in SEQ ID NO:33 (INAMG), CDR2 includes the amino acid sequence set forth in SEQ ID NO:39 (AITSGGSTNYADSVKG), and CDR3 includes the amino acid sequence set forth in SEQ ID NO:40 (DLYGEDYWI).

[0148] In some embodiments, the CDRs are numbered according to the Abm numbering method. In some embodiments, the CDRs are numbered according to the Chothia numbering method. In some embodiments, the CDRs are numbered according to the Kabat numbering method.

[0149] In some embodiments, CDR1 includes the amino acid sequence set forth in SEQ ID NO:41 (INAMX1), where X1 is G or A. In some embodiments, CDR2 includes 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 includes 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 includes the amino acid sequence set forth in SEQ ID NO:44 (DX1YGSDYWX2), where X1 is E or L, and X2 is D or I.

[0150] In some embodiments, the reagent is a single-domain antibody. In some embodiments, the reagent is a VHH antibody. In some embodiments, the reagent is a camelid antibody. In some embodiments, the camelid is a llama. In some embodiments, the reagent does not include other CDRs except for the CDRs described above.

[0151] In some embodiments, the reagent comprises a sequence EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTY YADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVT VSS (SEQ ID NO:30) that comprises and / or consists of the following.

[0152] In some embodiments, the reagent comprises a sequence EVQLVESGGGLVQAGGSLRLSCAASGSLFSINAMAWYRQAPGKQRELVAAITSSGSTNY ANSVKGRFTVSRDNAKNTMYLQMNSLKPEDTAVYYCVVDEYGSDYWIWGQGTQVTV SS (SEQ ID NO:31) that comprises and / or consists of the following.

[0153] In some embodiments, the reagent comprises a sequence QVQLVESGGGLVQAGGSLRLSCAASGSIFSINAMGWYRQAPGKQRERVAAITSGGSTN YADSVKGRFTISRDNAKNTVYLQMNNLEPRDAGVYYCVVDLYGEDYWIWGQGTQVT VSS (SEQ ID NO:32) that comprises and / or consists of the following.

[0154] 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 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 alanine residues. Each possibility represents a separate embodiment of the invention. In some embodiments, the alanine repeat linker consists of 3 alanine residues. In some embodiments, the His-tag is six His-tags.

[0155] In some embodiments, VHH sequences that specifically bind to the stem region of human CD28 and include a His tag were found to be: EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTY YADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVT VSSAAAHHHHHH (SEQ ID NO:45, clone 2A1); EVQLVESGGGLVQAGGSLRLSCAASGSLFSINAMAWYRQAPGKQRELVAAITSSGSTNY ANSVKGRFTVSRDNAKNTMYLQMNSLKPEDTAVYYCVVDEYGSDYWIWGQGTQVTV SSAAAHHHHHH (SEQ ID NO:46, clone 4A4); and QVQLVESGGGLVQAGGSLRLSCAASGSIFSINAMGWYRQAPGKQRERVAAITSGGSTN YADSVKGRFTISRDNAKNTVYLQMNNLEPRDAGVYYCVVDLYGEDYWIWGQGTQVT VSSAAAHHHHHH (SEQ ID NO:47, clone 4A1).

[0156] On the other hand, a reagent is provided that includes three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein: CDR-H1 includes the amino acid sequence set forth in SEQ ID NO:11 (GYTLTNY), CDR-H2 includes the amino acid sequence set forth in SEQ ID NO:12 (NTYTGK), CDR-H3 includes the amino acid sequence set forth in SEQ ID NO:13 (GDANQQFAY), CDR-L1 includes the amino acid sequence set forth in SEQ ID NO:14 (KASQDINSYLS), CDR-L2 includes the amino acid sequence set forth in SEQ ID NO:15 (RANRLVD), and CDR-L3 includes the amino acid sequence set forth in SEQ ID NO:16 (LQYDEFPPT). This antibody is referred to herein as M9.

[0157] In some embodiments, the reagent 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).

[0158] In some embodiments, the reagent comprises a heavy chain comprising the following amino acid sequence: DVKLVESGGGLVKLGGSLKLSCVASGFTFSSYYMSWVRQTPEKRLEWVATISDGGDNTYYAGTVTGRFTISRDFAKNTLYLQMNSLTSEDTAVYYCARIHWPYYFDSWGQGTTLTVSS (SEQ ID NO:23). In some embodiments, the variable region of the heavy chain comprises and / or consists of SEQ ID NO:23. In some embodiments, the reagent comprises a heavy chain comprising a polypeptide encoded by the following nucleic acid sequence: GACGTGAAGCTCGTGGAGTCTGGGGGAGGCTTAGTGAAGCTTGGAGGGTCCCTGAAACTCTCCTGTGTAGCCTCTGGATTCACTTTCAGTAGCTATTACATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCGACCATAAGTGATGGTGGTGATAACACCTACTACGCAGGCACTGTGACGGGCCGATTCACCATCTCCAGAGACTTTGCCAAGAACACCCTGTACCTGCAAATGAACAGTCTGACCTCTGAGGACACAGCCGTGTATTACTGTGCAAGAATTCATTGGCCTTACTATTTTGACTCCTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO:24). In some embodiments, the heavy chain consists of SEQ ID NO:24. Antibody M9 was sequenced and found to have a heavy chain consisting of SEQ ID NO:24. The CDRs of this heavy chain as determined using the Chothia scheme are SEQ ID NOs: 17-19.

[0159] In some embodiments, the reagent comprises a light chain comprising the following amino acid sequence: QFVLSQSPAILSASPGEMLTMTCRASSSVSYMNWYQQKPGSSPKPWIYATSDLASGVPA RFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSHPPTFGGGTKLEIR (SEQ ID NO:25). In some embodiments, the variable region of the light chain comprises or consists of SEQ ID NO:25. In some embodiments, the reagent comprises a light chain comprising a polypeptide encoded by the following nucleic acid sequence: CAATTTGTTCTCTCCCAGTCTCCAGCAATCCTGTCTGCATCTCCCGGGGAGATGCTCACAATGACTTGCAGGGCCAGCTCAAGTGTAAGTTATATGAACTGGTATCAGCAGAAGCCAGGATCTTCCCCCAAACCCTGGATTTATGCCACATCCGACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTATTCTCTCACAATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTCACCCACCCACGTTCGGAGGGGGGACCAAGCTGGAAATAAGA (SEQ ID NO:26). In some embodiments, the light chain consists of SEQ ID NO:26. Antibody M9 was sequenced and found to have a light chain consisting of SEQ ID NO:26. The CDRs of this light chain as determined using the Chothia scheme are SEQ ID NO:20-22.

[0160] In some embodiments, the reagent binds as a monomer. In some embodiments, the reagent binds as a dimer. In some embodiments, the reagent binds as a monomer and / or a dimer. In some embodiments, the reagent binds as a dimer but does not crosslink and / or activate mCD28. In some embodiments, the reagent binds as a dimer but binds only a single molecule of CD28. In some embodiments, the reagent binds monomeric CD28. In some embodiments, the reagent binds dimeric CD28. In some embodiments, the reagent binds monomeric and / or dimeric CD28.

[0161] In some embodiments, the reagent is not a CD28 agonist. In some embodiments, the reagent is not a CD28 antagonist. In some embodiments, the reagent is neither a CD28 agonist nor an antagonist.

[0162] The term "agonist" generally refers to a molecule, compound, or reagent that binds to a receptor and fully or partially activates the receptor. In some embodiments, the agonist binds at the same site as the natural ligand. In some embodiments, the agonist binds at an allosteric site different from the binding site of the natural ligand. The term "antagonist" generally refers to a molecule, compound, or reagent that binds to the receptor at the same site as the agonist or at another site, does not activate the receptor, and performs one or more of the following operations: interfering with or blocking the activation of the receptor by the natural ligand, and interfering with or blocking the activation of the receptor by a receptor agonist. In some embodiments, the antibodies of the present invention bind to mCD28 but do not activate or block the activation of the receptor. In some embodiments, they do not block the activation through CD86. In some embodiments, the antibodies of the present invention do not bind mCD28.

[0163] As used herein, a "direct agonist / antagonist" refers to a molecule that binds to a receptor (mCD28) and increases / decreases the signal transduction of that molecule through binding. In the case of mCD28, an agonist will bind to mCD28 and increase the mCD28 signal transduction in cells through binding. In some embodiments, the agonist increases T cell activation. In some embodiments, the agonist increases T cell proliferation. In some embodiments, the agonist increases the secretion of pro-inflammatory cytokines. Pro-inflammatory cytokines are well known in the art and are known to be secreted by activated T cells. Examples of pro-inflammatory cytokines include, but are not limited to, TNFα, IFNγ, IL-1B, IL-2, and IL-6. In some embodiments, the pro-inflammatory cytokine is IFNγ. In some embodiments, the pro-inflammatory cytokine is IL-2. In the case of mCD28, an antagonist will bind to mCD28 and decrease the mCD28 signal transduction in cells through binding. In some embodiments, the antagonist reduces T cell activation, reduces T cell proliferation, and / or reduces the secretion of pro-inflammatory cytokines. Molecules that affect receptor signal transduction to alter receptor signal transduction by contacting its ligand, contacting an inhibitor, contacting a co-receptor, or contacting any molecule other than the receptor being discussed are not considered direct agonists / antagonists. In some embodiments, the reagent of the present invention contacts sCD28 in serum and thereby allows an increase in the signal transduction through mCD28 on the cell. Although the result is an increase in mCD28 signal transduction, the antibody is not an mCD28 agonist or a direct agonist because its binding to mCD28 does not increase receptor signal transduction.

[0164] In some embodiments, the reagent does not bind to the ligand-binding domain of mCD28. In some embodiments, the reagent does not conceal or block access to the ligand-binding domain. In some embodiments, the reagent does not bind to, conceal, 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 following amino acid sequence: MLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKG (SEQ ID NO:8). In some embodiments, the reagent does not inhibit the binding of sCD28 to a ligand. In some embodiments, the CD28 ligand is selected from: CD80, CD86, and ICOS-L. In some embodiments, the CD28 ligand is CD86. In some embodiments, the CD28 ligand is CD80. In some embodiments, the CD28 ligand is ICOS-L. In some embodiments, CD86 is CD86-Fc. In some embodiments, CD80 is CD80-Fc.

[0165] In some embodiments, the reagent binds to the stalk region of CD28. In some embodiments, the reagent 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 reagent binds to monomeric sCD28. In some embodiments, the reagent binds to dimeric sCD28. In some embodiments, the reagent binds to monomeric sCD28, dimeric sCD28, or both. In some embodiments, the reagent binds to monomeric CD28 but not to dimeric CD28. In some embodiments, a fragment of the CD28 extracellular domain is the stalk region. In some embodiments, the reagent bound to CD28 prevents cleavage of CD28. In some embodiments, the reagent bound to CD28 prevents CD28 from shedding from the cell.

[0166] In some embodiments, the reagent binds to the cleavage site in the stem region. In some embodiments, the reagent binds at the cleavage site within mCD28. In some embodiments, the reagent binds at the cleavage site of at least one protease. In some embodiments, the reagent binds at the cleavage site of MMP-2.

[0167] In some embodiments, the reagent does not bind to the ligand-binding domain of mCD28. In some embodiments, the reagent does not conceal or block access to the ligand-binding domain. In some embodiments, the reagent binds at the cleavage site. In some embodiments, the reagent conceals, occludes or blocks access to the cleavage site. In some embodiments, the reagent binds, blocks, occludes or conceals the protease cleavage site. In some embodiments, the reagent does not bind to the protease cleavage site but occludes the site. In some embodiments, the reagent blocks access to the protease cleavage site. In some embodiments, the reagent creates steric hindrance that blocks the protease cleavage site. In some embodiments, the reagent does not bind to the protease cleavage site, but the binding of the reagent creates a conformational change in mCD28 that blocks the protease cleavage site. In some embodiments, the binding of the reagent 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 reagent blocks protease access to the cleavage site. In some embodiments, the reagent binds to PSPL in the stem domain of mCD28.

[0168] 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.

[0169] In some embodiments, the reagent does not bind to the stem region of CD28 having a mutated cleavage site. In some embodiments, the stem region of CD28 having a mutated cleavage site is not a substrate for a protease. In some embodiments, the stem region of CD28 having a mutated cleavage site is not a substrate for a metalloprotease. In some embodiments, the stem region of CD28 having a mutated cleavage site is not a substrate for a matrix metalloprotease. In some embodiments, the stem region of CD28 having a mutated cleavage site is not a substrate for matrix metalloprotease 2 (MMP-2). In some embodiments, the stem 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 of SEQ ID NO:1. In some embodiments, the mutated cleavage site is an amino acid substitution of leucine 145 of SEQ ID NO:1. In some embodiments, the amino acid substitution of leucine 145 of SEQ ID NO:1 is lysine.

[0170] In some embodiments, the reagent does not modulate CD28 function and / or signaling. In some embodiments, the reagent does not degrade mCD28. In some embodiments, the reagent does not cause or promote mCD28 degradation. In some embodiments, the signaling is mCD28-mediated immune cell activation. In some embodiments, the reagent does not inhibit immune cell activation. In some embodiments, the reagent does not induce CD28 receptor internalization or recycling. Co-stimulation through mCD28 is crucial for the immune activation of T cells. Protease cleavage removes the ligand-binding domain in the extracellular region of CD28 from the transmembrane and cytoplasmic portions of the protein remaining in the membrane. Thus, the cleaved CD28 cannot conduct signals and cannot contribute to T cell activation. Thus, a reagent that blocks cleavage and is also an antagonist does not allow mCD28 activation. Similarly, a reagent that blocks cleavage but is also an agonist can induce abnormal T cell activation and potentially an autoimmune response.

[0171] In some embodiments, the reagent does not reduce the surface level of mCD28 on immune cells. In some embodiments, the immune cells are T cells. In some embodiments, the reagent reduces the surface level of mCD28 by less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 7%, 5%, 3%, 2%, or 1%. Each possibility represents a separate embodiment of the invention.

[0172] In some embodiments, the binding of the reagent to the cell does not kill the cell. In some embodiments, the binding of the reagent to the cell does not cause cell death. In some embodiments, the reagent does not induce antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the reagent does not induce complement-dependent cytotoxicity (CDC). In some embodiments, the reagent does not induce ADCC and / or CDC. In some embodiments, the reagent is an antibody and 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 IgG1 or IgG3, which is mutated to reduce cell death mediated by binding of the antibody. In some embodiments, the mutation mutates the Fc receptor-binding domain. In some embodiments, the Fc domain of the antibody is engineered or mutated to reduce CDC, ADCC, or both. Fc engineering is well known in the art and any mutation or amino acid change known to reduce antibody-mediated cell killing can be used.

[0173] In some embodiments, the reagent lacks an Fc domain. In some embodiments, the reagent is an antigen-binding domain that lacks an Fc domain. In some embodiments, the reagent is a single-domain antibody. In some embodiments, the reagent is a camelid antibody, a shark antibody, or a nanobody.

[0174] In some embodiments, the reagent is a non-antibody protein. In some embodiments, the reagent is a small molecule. In some embodiments, the reagent is a nucleic acid molecule. In some embodiments, the reagent is a synthetic peptide. In some embodiments, the reagent is a synthetic binding protein. In some embodiments, the synthetic peptide is based on a non-antibody scaffold. In some embodiments, the reagent is an antibody mimetic. In some embodiments, the molar mass of the antibody mimetic is less than 100, 90, 80, 70, 60, 50, 40, 30 or 20 kDa. Each possibility represents a separate embodiment of the invention. In some embodiments, the reagent 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, alpha antibodies, anticalins, avimers, DARPins, fynomers, Kunitz domain peptides, monobodies, and nanoCLAMPS. In some embodiments, the antibody mimetic is a DARPin.

[0175] In some embodiments, the reagent inhibits protease cleavage by at least one protease. In some embodiments, the protease is a metalloprotease. In some embodiments, the protease is a matrix metalloprotease. In some embodiments, the protease is a serine protease. In some embodiments, the protease is a cysteine protease. In some embodiments, the protease is a threonine protease. In some embodiments, the protease is a serine, cysteine, or threonine protease. In some embodiments, the protease is an aspartic protease. In some embodiments, the protease is a glutamic protease. In some embodiments, the protease is selected from aspartic, glutamic, serine, cysteine, and threonine proteases. In some embodiments, the protease is an asparaginyl 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-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.

[0176] Methods of use

[0177] In another aspect, provided is a method of treating and / or preventing cancer in a subject in need thereof, the method comprising administering a reagent of the present invention.

[0178] In another aspect, provided is a method of improving immunotherapy in a subject in need thereof, the method comprising administering a reagent of the present invention.

[0179] In another aspect, provided is a method of reducing sCD28 in a subject in need thereof, the method comprising administering a reagent of the present invention.

[0180] In some embodiments, the immunotherapy is a 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 blocking a PD-1 checkpoint. In some embodiments, the immunotherapy comprises administering allogeneic, syngeneic or autologous immune cells to a subject. In some embodiments, the immune cells are T cells. In some embodiments, the subject in need of immunotherapy has cancer. In some embodiments, the subject has cancer. In some embodiments, the cancer is an sCD28-positive cancer. In some embodiments, the cancer is an sCD28-high cancer. In some embodiments, the subject is at risk of developing cancer.

[0181] As used herein, the term "treatment" of a disease, disorder or condition includes the alleviation of at least one symptom thereof, the reduction of its severity, or the inhibition of its progression. Treatment does not necessarily mean the complete cure of the disease, disorder or condition. As an effective treatment, the compositions useful herein only need to reduce the severity of the disease, disorder or condition, reduce the severity of the symptoms associated therewith, or improve the quality of life of the patient or subject.

[0182] In some embodiments, reducing comprises administering to a subject at least one reagent of the present invention. As used herein, the terms "administering" and similar terms refer to any method of delivering a composition containing an active agent to a subject in a manner that provides a therapeutic effect in reasonable medical practice. One aspect of the subject matter provides oral administration of a therapeutically effective amount of a reagent of the present invention to a patient in need thereof. Other suitable routes of administration may include parenteral, subcutaneous, intravenous, intramuscular or intraperitoneal.

[0183] By another aspect, there is provided a pharmaceutical composition comprising a reagent of the present invention and a pharmaceutically acceptable carrier, adjuvant or excipient. In some embodiments, the administration is using the pharmaceutical composition of the present invention.

[0184] As used herein, the terms "carrier", "excipient" or "adjuvant" refer to any component of a pharmaceutical composition that is not an active agent. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material, any type of formulation aid, or merely a sterile aqueous medium such as saline. Some examples of materials that can serve as pharmaceutically acceptable carriers are sugars (such as lactose, glucose and sucrose), starches (such as corn starch and potato starch), celluloses and their derivatives (such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate); acacia powder; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil, etc.; polyols such as glycerol, sorbitol, mannitol, polyethylene glycol, etc.; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide, aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethanol and phosphate buffer solutions, and other non-toxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of substances that can be used as carriers herein include sugars, starches, celluloses and their derivatives, powdered acacia, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffer solutions, cocoa butter (suppository base), emulsifying agents and other non-toxic pharmaceutically compatible substances used in other pharmaceutical formulations. There may also be wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, excipients, stabilizers, antioxidants and preservatives. Any non-toxic, inert and effective carrier can be used to formulate the compositions contemplated herein. Suitable pharmaceutically acceptable carriers, excipients and diluents in this regard are well known to those skilled in the art, such as those described in The Merck Index, 13th Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, N.J. (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, 10th Edition (2004); and the "Inactive Ingredient Guide," U.S. Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, the entire contents of which are hereby incorporated by reference in their entirety.Examples of pharmaceutically acceptable excipients, carriers, and diluents that can be used in the present compositions include distilled water, physiological saline, Ringer's solution, dextrose solution, Hank's solution, and DMSO. These additional inactive ingredients, as well as effective formulations and administration procedures, are well known in the art and 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 presently described compositions can also be included in artificially generated structures, such as liposomes, ISCOMS, slow release granules, and other carriers that increase the half-life of a peptide or polypeptide in serum. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. Liposomes used with the presently described peptides are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and sterols, such as cholesterol. The choice of lipids is generally determined by considerations such as liposome size and stability in the blood. A variety of methods can be used to prepare liposomes, for example, Coligan, J.E. et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, and also see U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0185] The carrier can total from about 0.1% to about 99.99999% of the weight of the pharmaceutical composition presented herein.

[0186] In some embodiments, the methods of the invention do not degrade or cause degradation of mCD28. In some embodiments, the methods of the invention do not reduce the level of mCD28 on immune cells. In some embodiments, the methods of the invention do not reduce mCD28-mediated immune cell activation. In some embodiments, the methods of the invention maintain the level of mCD28 on immune cells in a subject. In some embodiments, the methods of the invention increase the level of mCD28 on immune cells in a subject.

[0187] In some embodiments, the reduction is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% reduction of sCD28. Each possibility represents a separate embodiment of the invention. In some embodiments, the reduction is in terms of serum sCD28. In some embodiments, the reduction is in terms of the blood level of sCD28. In some embodiments, the reduction is in terms of the level of sCD28 in the tumor microenvironment (TME).

[0188] In some embodiments, the subject's blood comprises elevated sCD28 levels. In some embodiments, prior to reduction, the subject's blood comprises elevated sCD28 levels. In some embodiments, the levels are elevated above those of a healthy subject. 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 the levels of a healthy subject. Each possibility represents a separate embodiment of the invention. In some embodiments, the levels are elevated above 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45, or 50 ng / ml of blood. Each possibility represents a separate embodiment of the invention. In some embodiments, the levels are elevated above 5 ng / ml. In some embodiments, the levels are elevated above 10 ng / ml. In some embodiments, the levels are elevated above 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 / ml of blood. Each possibility represents a separate embodiment of the invention. In some embodiments, prior to reduction, 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 / ml of blood. Each possibility represents a separate embodiment of the invention. In some embodiments, the subject's blood comprises at least 5 ng / ml of sCD28. In some embodiments, the subject's blood comprises at least 10 ng / ml of sCD28. In some embodiments, the subject's blood comprises at least 20 ng / ml of sCD28. In some embodiments, prior to reduction, the subject's blood comprises at least 5 ng / ml of sCD28. In some embodiments, prior to reduction, the subject's blood comprises at least 10 ng / ml of sCD28. In some embodiments, prior to reduction, the subject's blood comprises at least 20 ng / ml of sCD28.

[0189] In some embodiments, the subject has cancer. In some embodiments, the cancer is a cancer treatable with PD-1 / PD-L1 therapy. In some embodiments, the subject has undergone PD-1 / PD-L1 therapy. In some embodiments, the subject is a non-responder to PD-1 / PD-L1 therapy. In some embodiments, the subject has not undergone PD-1 / PD-L1 therapy. In some embodiments, the methods of the invention are carried out in conjunction with PD-1 / PD-L1 therapy. In some embodiments, the methods of the invention are carried out prior to PD-1 / PD-L1 therapy.

[0190] In some embodiments, the method further comprises administering to the subject another immunotherapy. In some embodiments, the method further comprises administering an immunotherapy based on PD-1 and / or PD-L1. In some embodiments, the other immunotherapy is a 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 CAR-T. In some embodiments, the CAR is CAR-NK. In some embodiments, the other immunotherapy is a cancer vaccine.

[0191] As used herein, the terms "CAR-T cells" and "CAR-NK cells" refer to engineered receptors that are specific for at least one target protein (e.g., an immunogenic protein having increased expression after contact 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.

[0192] CAR-T and CAR-NK cells and their vectors are well known in the art. Such cells target receptor-bound proteins and are cytotoxic to them. In some embodiments, the CAR-T or CAR-NK cells target at least one viral protein. In some embodiments, the CAR-T or CAR-NK cells target multiple viral proteins. In some embodiments, the CAR-T or CAR-NK cells target viral proteins having increased expression due to contact with an epigenetic modifier.

[0193] The construction of CAR-T cells is well known in the art. In a non-limiting example, monoclonal antibodies against viral proteins can be produced, and then vectors encoding the antibodies are constructed. The vectors will also include co-stimulatory signal regions. In some embodiments, the co-stimulatory 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 the ligand that specifically binds to CD83. In some embodiments, the vector further includes a CD3Z signaling domain. Then the vector is transfected into T cells, for example, by lentiviral infection.

[0194] In some embodiments, the cancer is a cancer with sCD28 levels. In some embodiments, the cancer includes high sCD28 levels. In some embodiments, the elevated and / or high sCD28 levels are at and / or above levels of 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 includes high sCD28 levels. In some embodiments, the 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.

[0195] Kits

[0196] On the other hand, a kit including at least one reagent of the present invention or the pharmaceutical composition of the present invention is provided.

[0197] In some embodiments, the kit further comprises an immunotherapeutic agent based on PD-1 and / or PD-L1. In some embodiments, the kit comprises a label indicating the use of the reagent of the present invention in combination with an immunotherapeutic agent based on PD-1 and / or PD-L1. In some embodiments, the kit comprises a label indicating the use of a therapeutic agent based on PD-1 and / or PD-L1 in combination with the antibody or pharmaceutical composition of the present invention.

[0198] In another aspect, there is provided a kit comprising an immunotherapeutic agent based on PD-1 and / or PD-L1, which comprises a label indicating the use of a therapeutic agent based on PD-1 and / or PD-L1 in combination with the antibody or pharmaceutical composition of the present invention.

[0199] In some embodiments, the kit of the present invention is used for treating cancer. In some embodiments, the kit of the present invention is a diagnostic kit. In some embodiments, the kit of the present invention is used to determine the serum level of sCD28 in a subject in need thereof. In some embodiments, the subject has cancer. In some embodiments, the kit of the present invention is used to determine the suitability of a subject to be treated with the reagent or pharmaceutical composition of the present invention. In some embodiments, the kit is used to determine the suitability of a subject to be treated with an anti-PD-1 / PD-L1 based immunotherapy.

[0200] Methods for producing the reagents

[0201] In another aspect, there is provided a method for producing a reagent that inhibits proteolytic cleavage of mCD28 on the surface of cells, which comprises:

[0202] a. obtaining a reagent that binds to the extracellular domain of CD28 or a fragment thereof, wherein the reagent is less than 100 kDa;

[0203] b. testing the binding of the obtained reagent to mCD28 on the cell surface; and

[0204] c. selecting a reagent that binds to cell surface mCD28;

[0205] Thereby producing a reagent that inhibits proteolytic cleavage of mCD28 on the surface of cells.

[0206] In another aspect, there is provided a method for producing a reagent that inhibits proteolytic cleavage of mCD28 on the surface of cells, which comprises:

[0207] d. culturing a host cell comprising one or more vectors, the vectors comprising a nucleic acid sequence encoding the reagent, wherein the nucleic acid sequence is the nucleic acid sequence of a reagent selected by:

[0208] i. Obtain a reagent that binds to the extracellular domain of CD28 or a fragment thereof, wherein the reagent is less than 100 kDa;

[0209] ii. Test the binding of the obtained reagent to mCD28 on the cell surface; and

[0210] iii. Select a reagent that binds to cell surface mCD28;

[0211] Thereby generating a reagent that inhibits the proteolytic cleavage of mCD28 on the surface of cells.

[0212] In some embodiments, the method further comprises testing the ability to block the proteolytic cleavage of mCD28 on the cell surface. In some embodiments, the reagent is an anti-cleavage agent. In some embodiments, the reagent is an anti-shedding agent. In some embodiments, the reagent reduces the shedding of sCD28 in a subject. In some embodiments, the reagent reduces the cleavage of mCD28. In some embodiments, the reagent reduces the cleavage of mCD28 in a subject.

[0213] 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.

[0214] As used herein, the term "extracellular domain of CD28" refers to the N-terminal portion of CD28 that precedes 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 includes the CD28 stalk domain. In some embodiments, the extracellular domain of CD28 comprises the following sequence or consists of: 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.

[0215] As used herein, "fragment" refers to a partial polypeptide that forms part 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 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 invention. In some embodiments, a reagent that obtains a fragment of the extracellular domain that binds CD28 is a reagent that specifically binds to the CD28 stalk domain.

[0216] In some embodiments, the method further comprises assaying mCD28 downstream signaling in the presence of the obtained reagent and selecting at least one reagent that neither substantially agonizes nor substantially antagonizes mCD28 signaling. In some embodiments, the selection is selecting at least one reagent that does not antagonize mCD28 signaling. Those skilled in the art will understand that for cancer treatment, agonizing CD28 signaling may not be harmful, but antagonizing the signaling would be counterproductive.

[0217] In some embodiments, the ability of the test reagent to block cleavage comprises measuring sCD28 in the serum of activated immune cells in the presence or absence of the reagent. In some embodiments, the ability of the test reagent to block cleavage comprises mixing the reagent, a protease, and the extracellular domain of CD28 or a fragment thereof that comprises a cleavage site. In some embodiments, the test further comprises sequencing the extracellular domain of CD28 or a fragment thereof to examine truncation and / or cleavage. In some embodiments, the test 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, the test further comprises measuring the production of sCD28 from cells expressing mCD28 in the presence of the reagent and the protease.

[0218] In some embodiments, obtaining the reagent comprises immunizing a shark or a camelid with the CD28 extracellular domain or a fragment thereof and collecting antibodies from the immunized organism. In some embodiments, obtaining the reagent comprises screening a library of reagents that bind to the CD28 extracellular domain or a fragment thereof and selecting the binding reagents.

[0219] In some embodiments, collecting the antibody comprises extracting B cells from the spleen of an immunized shark or camelid. In some embodiments, the B cells are fused with melanocytes to produce hybridomas. In some embodiments, the antibody is collected from the culture medium of the hybridomas. In some embodiments, obtaining the reagent comprises immunizing an organism with the extracellular domain of CD28 or a fragment thereof and collecting the antibody from the immunized organism. In some embodiments, the organism is a mouse. In some embodiments, the organism is selected from rabbits, mice, rats, sharks, camelids, chickens, goats, and phages. In some embodiments, the camelid is selected from camels and llamas. In some embodiments, collecting comprises drawing blood. In some embodiments, collecting comprises:

[0220] e. extracting B cells from the spleen of the immunized organism;

[0221] f. fusing the extracted B cells with myeloma cells to produce hybridomas; and

[0222] g. collecting the antibody from the hybridomas.

[0223] In some embodiments, obtaining the reagent comprises screening a library of reagents that bind to the extracellular domain of CD28 or a fragment thereof and selecting the reagents that so bind. In some embodiments, the library is a phage display library. In some embodiments, the library is an immune 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 VHH antibody library. In some embodiments, the library is a single-chain, single-domain, or nanobody library. In some embodiments, obtaining the reagent comprises sequencing the reagent. In some embodiments, obtaining the reagent comprises producing a recombinant form of the reagent. In some embodiments, selecting the reagent comprises sequencing the reagent. In some embodiments, selecting the reagent comprises producing a recombinant form of the reagent. In some embodiments, the recombinant form is produced from the sequence of the reagent. In some embodiments, the method further comprises humanizing the reagent.

[0224] Expression of a nucleic acid molecule encoding a reagent intracellularly is well known to those skilled in the art. It can be carried out by many methods such as transfection, viral infection, or direct alteration of the cell genome. In some embodiments, the gene is present 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.

[0225] The vector nucleic acid sequence generally comprises at least an origin of replication for propagation in a cell and optionally additional elements such as a heterologous polynucleotide sequence, expression control elements (e.g., promoters, enhancers), selectable markers (e.g., antibiotic resistance), polyadenylation sequences.

[0226] The vector can be a DNA plasmid delivered by a non-viral method or a viral method. The viral vector can be a retroviral vector, a herpesvirus vector, an adenovirus vector, an adeno-associated virus vector, or a poxvirus vector. The promoter can be active in mammalian cells. The promoter can be a viral promoter.

[0227] In some embodiments, the nucleic acid sequence encoding the reagent is operably linked to the promoter. The term "operably linked" is intended to mean that the target nucleotide sequence is linked to one or more regulatory elements in a manner that permits the 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).

[0228] In some embodiments, the vector is introduced into the cell by standard methods, including electroporation (e.g., as described in From et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985)), heat shock, viral vector infection, high-velocity ballistic penetration of small particles with nucleic acids within or on the surface of beads or particles (Klein et al., Nature 327, 70-73 (1987)) and / or similar methods.

[0229] As used herein, the term "promoter" refers to a set of transcriptional control modules that cluster around the start site of RNA polymerase, i.e., RNA polymerase II. The promoter consists of discrete functional modules, each module consisting of about 7-20 bp of DNA and containing recognition sites for one or more transcriptional activators or repressor proteins.

[0230] In some embodiments, the transcription sequence is transcribed by RNA polymerase II (RNAP II and Pol II). RNAP II is an enzyme present in eukaryotic cells. It catalyzes the transcription of DNA to synthesize mRNA and the precursors of most snRNA and microRNA.

[0231] In some embodiments, mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(±), pGL3, pZeoSV2(±), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81, which are available from Invitrogen; pCI, which is available from Promega; pMbac, pPbac, pBK-RSV and pBK-CMV, which are available from Strategene; pTRES, which is available from Clontech, and their derivatives.

[0232] In some embodiments, the present invention uses expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine 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 expression of a protein under the direction of an SV-40 early promoter, an SV-40 late promoter, a metallothionein promoter, a murine mammary tumor virus promoter, a Rous sarcoma virus promoter, a polyhedrin promoter, or any other promoter that is effective for expression in eukaryotic cells.

[0233] In some embodiments, recombinant viral vectors, which have advantages such as lateral infection and targeting specificity, are used for in vivo expression. In one embodiment, lateral infection, which is, for example, inherent in the life cycle of retroviruses, is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. In one embodiment, the result is rapid infection of a large area, where most of the area was initially not infected by the original viral particles. In one embodiment, viral vectors that are unable to spread laterally are produced. In one embodiment, this feature may be useful if the desired goal is to introduce a specific gene into only a local number of target cells.

[0234] 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), 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 with recombinant viral vectors, liposome transfection, electroporation, and infection. Additionally, for positive-negative selection methods, see U.S. Patent Nos. 5,464,764 and 5,487,992.

[0235] It should be understood that, in addition to elements necessary for the transcription and translation of the inserted coding sequence (encoding a polypeptide), the expression constructs of the present invention can also include sequences engineered to optimize the stability, production, purification, yield, or activity of the expressed polypeptide.

[0236] In another aspect, there is provided a reagent produced by the method of the present invention.

[0237] In another aspect, there is provided a pharmaceutical composition comprising a reagent produced by the method of the present invention and a pharmaceutically acceptable carrier, excipient, or adjuvant.

[0238] As used herein, when combined with a value, the term "about" refers to plus or minus 10% of the reference value. For example, a length of about 1,000 nanometers (nm) refers to a length of 1,000 ± 100 nm.

[0239] Note that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides, and reference to "a polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. Further note that the claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as antecedent basis for the use of exclusive terms, such as "solely", "only", and the like, or the use of "negative" limitations associated with the recitation of claim elements.

[0240] In those instances where a convention such as "at least one of A, B, and C, etc." is used, generally, such constructions are intended in the sense that would be understood by one of ordinary skill in the art (e.g., "a system having at least one of A, B, and C" would include, without limitation, a system having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). One of ordinary skill in the art will further understand that, in the specification, claims, or drawings, substantially any disjunctive word and / or phrase presenting two or more alternative terms should be understood as contemplating the possibility of including one of the terms, either term, or both terms. For example, the phrase "A or B" will be understood to include the possibility of "A" or "B" or "A and B".

[0241] It should be understood that, for clarity, certain features of the invention described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, the various features of the invention described in the context of a single embodiment may also be provided separately or in any suitable sub-combination. All combinations of embodiments relevant to the invention are specifically included in the invention and are disclosed herein as if each combination were individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and their elements are specifically included in the invention and are disclosed herein as if each such sub-combination were individually and explicitly disclosed herein.

[0242] Other objects, advantages, and novel features of the invention will become apparent to those of ordinary skill in the art upon examination of the following examples, which are not intended to be limiting. In addition, each of the various embodiments and aspects of the invention as described above and claimed in the claims section finds experimental support in the following examples.

[0243] The various embodiments and aspects of the invention as described above and claimed in the claims section find experimental support in the following examples.

[0244] Embodiment

[0245] Generally, the nomenclature used herein and the laboratory procedures used in this invention include molecular, biochemical, microbial, and recombinant DNA techniques. These techniques are explained in detail in the literature. See, for example, "Molecular Cloning: A Laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R.M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); the methods described in U.S. Patent Nos. 4,666,828, 4,683,202, 4,801,531, 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J.E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-IIIColigan J.E., ed. (1994); Stites et al.(eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated herein by reference. Other general references are also provided in this document.

[0246] Materials and Methods

[0247] Antibodies - Commercially available mouse monoclonal anti - CD28 clone #CD28.2 (Biolegend, Cat. No. 302902) and FITC - conjugated (Biolegend, Cat. No. 302906). Goat polyclonal anti - CD28 (R&D system, Cat. No. AF - 342 - PB). FITC - conjugated anti - human PD - L1 (BD bioscience, Cat. No. 558065). APC - conjugated anti - human PD - L2 (Biolegend, Cat. No. 345508). PE - conjugated anti - human IDO (R&D system, Cat. No. IC6030P). Goat anti - mouse IgG Alexa Fluor 647 (Biolegend, Cat. No. 405322). Donkey anti - human IgG (H + L) Alexa Fluor 647 (Jackson immune research, Cat. No. 709 - 605 - 149). Goat anti - mouse IgG HRP (Jackson immune research, Cat. No. 115 - 035 - 071). Anti - human CD3 clone OKT3 (Biolegend, Cat. No. 317304). Anti - human PD - 1 pembrolizumab (MK - 3475). Human IgG (Sigma, Cat. No. I4506).

[0248] Isolation of VHH targeting the stalk region of the human CD28 receptor - The genetic code of peripheral blood B cells, derived from a non-immunized Llama used for the first experiment, was used to construct a phage library consisting of particles expressing individual VHHs as fusion proteins with a C-terminal His6-Myc tag. The library used for the first experiment was used to select nanobodies with the ability to bind to the stalk region of human CD28. The biotinylated recombinant CD28-Fc chimera or the oxidized dimer peptide with the sequence "HVKGKHLCPSPLFPGPSKP (SEQ ID NO:10)" with biotin addition at the C-terminus was screened. Each antigen was bound to streptavidin magnetic beads, which were blocked with skim milk. The same antigen was used in three consecutive selection rounds, varying the phage input and antigen concentration, for selection of phages in solution. The blocked beads without antigen were used as a control. Elution of the bound phages was performed with trypsin for 20 minutes. The enrichment rate during the solution selection process was calculated as the ratio between the number of phages eluted from the CD28 antigen selection conditions and the number of phages eluted from the no-antigen selection conditions. 279 individual phage monoclonal of the selected output, in phage or periplasmic form, were verified for antigen binding by ELISA and characterized for binding to membrane CD28 by flow cytometry. 72 clones showed specific binding to the stalk region peptide in periplasmic form, 22 were proven to have unique CDR sequences, while only 6 were found to belong to a unique CDR3 family. 6 VHHs were produced as recombinant proteins in CHO cells with a c-terminal His tag and evaluated for their anti-shedding activity and cell binding. Transfection - The CD28wt (encoding the full-length CD28 transcript) plasmid was generated by cloning the DNA sequence into the PCDNA3.1 vector. Transfection was performed using the Jet Pei transfection reagent (PolyPlus transfection). Stable transfectants were selected in medium containing G418.

[0249] ELISA- Commercial ELISA kits were used to quantify the amounts of interferon-γ (Biolegend, Cat. No. 430103), human interleukin 2 (Biolegend, Cat. No. 431802), human interleukin 6 (Biolegend, Cat. No. 430502), human interleukin 10 (Biolegend, Cat. No. 430603), human tumor growth factor β1 (Biolegend, Cat. No. 436708), human interleukin β1 (Biolegend, Cat. No. 437004) and human CD28 (R&D system, Cat. No. DY342). Cell proliferation and viability (MTT assay) (Roche, Cat. No. 11465007001) were performed according to the manufacturer's instructions. Kynurenine (IDO activity) ELISA kit (ImmuSmol, Cat. No. BA E-2200) was performed according to the manufacturer's instructions.

[0250] CD28 stalk region binding assay - Biotin-conjugated wild-type or L145K CD28 stem region dimer peptides were immobilized on neutravidin-coated ELISA maxi-sorb plates. VHH clones were serially diluted (0.2 - 5 μg / mL), and bound VHH was detected using an anti-His tag-HRP conjugated antibody and developed with TMB.

[0251] Cytokine multiplex - Multiple cytokines were simultaneously evaluated using ProcartaPlex (Invitrogen, Cat. No. PPX-07-MXXGPY2) on a Magpix system (Millipore).

[0252] Flow cytometry - Generally, cells were kept on ice during all steps. Before staining, 5 × 10 5 cells were blocked with 50 μg / mL human IgG (Sigma, Cat. No. I4506) in FACS buffer (PBS containing 0.1% BSA) for 15 min. Antibodies were used at the concentrations recommended by the manufacturer and incubated for 30 min in the dark. Incubation was performed in a 96-well U-bottom plate with a volume of 100 μL. Cells were washed twice with 200 μL of FACS buffer and transferred to a FACS tube in 150 μL of FACS buffer for analysis. Cells were analyzed on a Gallios flow cytometer (Beckman Coulter) using Kaluza for Gallios flow cytometer acquisition software.

[0253] Isolation of cell lines and human immune cells- Obtained the Jurkat leukemia T-cell lymphoblastoid cell line clone E6.1 and SCC-25 tongue squamous cell carcinoma from ATCC. PBMC were isolated from fresh blood samples of healthy donors using standard lymphocyte separation medium (MBP, Cat. No. 850494). CD3 cells were isolated from fresh blood samples of healthy donors using the RosetteSEP TM Human T Cell Enrichment Kit (STEMCELL, Cat. No. 15061) by negative selection method. CD4 cells were isolated from fresh blood samples of healthy donors using the RosetteSep TM Human T Cell Enrichment Kit (STEMCELL, Cat. No. 19059) by negative selection method. Monocytes were isolated from fresh blood samples of healthy donors using the RosetteSep TM Human T Cell Enrichment Kit (STEMCELL, Cat. No. 17952). All cells were grown in complete RPMI-1640 medium supplemented with 10% HI-FCS and pen / strep mixture.

[0254] CD86 - blocking FACS - At room temperature, 0.5×10 6 HEK293 cells stably transfected with human CD28 were incubated with 2 μg / mL CD86-Fc (R&D systems, Cat. No. 141-B2) with or without anti-CD28 antibody (CD28.2, 10 μg / mL) or VHH clone (30 μg / mL) for 30 min. The cells were washed and secondary binding was performed on ice for 20 min using fluorophore-conjugated anti-human heavy and light chain antibodies diluted 1:5000.

[0255] Dendritic cell differentiation - Monocytes were cultured at a density of 1×10 6 / mL in RPMI medium, and growth factors were supplemented on days 3 and 6. 50 ng / mL GM-CSF and 20 ng / mL IL-4 were used to induce immature dendritic cells (iDC) for 6 days. When needed, iDC were further differentiated into mature dendritic cells by adding 100 ng / mL LPS for 48 hours. The generated cell population was tested for the designated phenotype by FACS analysis of relevant markers and analysis of the secretion of characteristic cytokines.

[0256] Metalloproteinases- Use commercially available recombinant human metalloproteinase MMP-2 from Anaspec (Cat. No. AS-72005) or R&D system (Cat. No. 902-MP). Purchase commercially available recombinant human metalloproteinase MMP-13 from R&D system (Cat. No. 511-MM). Activate Pro-MMP2 and Pro-MMP-13 with 1 mM mercury aminophenylacetate (APMA) at 37 °C for 1-2 hours according to the manufacturer's protocol.

[0257] Protease inhibitors - Add protease inhibitors at the specified concentration at the start of each experiment. In the one-week trial, another portion of the inhibitor was added at the final concentration after 3 days. The protease inhibitors used were TAPI-1 (Cayman, Cat. No. 18505), GM6001 (Santa Cruz, Cat. No. SC-203979), TMI-1 (Sigma, Cat. No. PZ0336), and GI254023X (Sigma, Cat. No. SML0789). The protease mixture mentioned therein was composed of a mixture of TAPI-1 and GM6001 in an equimolar ratio.

[0258] Synthetic peptides - Design a substrate peptide in the final form of "DYKDDDDKGGGGGHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 41)-biotin" to include the amino acid sequence of the human CD28 stem region (His134-Pro152) between the N-terminal cMyc tag followed by a 5-glycine sequence and the C-terminal biotin conjugation. The peptide was custom synthesized by Gencust Europe. The cysteine residue at position 141 was used to generate a dimeric peptide via a disulfide bond. Similarly, a CD28 stem region peptide with a mutation at the cleavage site, a leucine-to-lysine substitution at position 145, was synthesized, which had the final form of "DYKDDDDKGGGGGHVKGKHLCPSPKFPGPSKP (SEQ ID NO: 42)-biotin".

[0259] In vitro cleavage assay- Purified recombinant MMP-2 or MMP-13 (50 ng) was incubated with 0.125 μM of a dimerized c-Myc-tagged and biotinylated substrate peptide in the presence or absence of an MMP inhibitor (TMI-1, 50 nM), M9 Fab, or the indicated VHH clones at various concentrations (0.4 - 10 μg / mL) for 5 h. 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 neutral avidin plate to bind the peptide. After incubation at room temperature for 1 h, the plate was washed and detection of the uncleaved peptide was performed using an anti-cMyc antibody conjugated to HRP.

[0260] PHA activation of CD4 T cells or Jurkat T cell lines for the production of soluble CD28 - 1×10 5 Jurkat cells or CD4 T cells were incubated with the indicated concentration of phytohemagglutinin (Sigma, Cat. No. L8902) and with various protease inhibitors for an additional 5 days (Jurkat) or 7 days (CD4 T cells).

[0261] SEB or CMV activation of PBMC for the production of soluble CD28 - 0.3×10 6 PBMCs were stimulated with 0.5 ng / mL SEB (Sigma, Cat. No. S4881) in the presence or absence of various protease inhibitors at the indicated concentrations in 48-well plates at 37 °C for 5 - 7 days. Optionally, 0.1×10 6 PBMCs were stimulated with 0.5 ng / mL SEB in 96-well plate format assays. For CMV stimulation, 0.5×10 6 PBMCs were activated with 0.5 μg / mL CMV peptivator (Milteny Biotec, Cat. No. 130 - 093 - 435) in the presence or absence of various protease inhibitors at the indicated concentrations in 96-well plates at 37 °C for 2 - 5 days. For the continuous shedding experiment, PBMCs were stimulated with SEB or CMV in 24-well plates for 24 h, the cells were taken and washed three times with RPMI without stimulant, and then replated in 96-well plates. Samples were taken at the indicated times and placed under freezing conditions until soluble CD28 was assayed.

[0262] Cell assays for evaluating the anti - shedding activity of VHH - For SEB activation of PBMCs, 0.1×10 6PBMC were incubated for 5 - 7 days. For PHA-activated T cells, 0.1×10 6 CD4 T cells were incubated for 5 - 7 days at 37°C in a 96-well plate with / without various treatments at specified concentrations with phytohemagglutinin (Sigma, Cat. No. L8902) at a specified concentration and 200 IU / mL of IL-2 (Proleukine). For the HEK spontaneous CD28 shedding assay, 0.1×10 5 HEK cells were incubated for 48 hours at 37°C in a 96-well plate with / without various treatments at specified concentrations.

[0263] Mixed lymphocyte reaction -1×10 5 immature DCs were mixed with 5×10 5 isolated autologous CD3 T cells for 6 days.

[0264] SEB or CMV stimulation assays with ectopically recombinant human CD28, human CTLA - 4, and human CD80 -For CMV stimulation, 0.5×10 6 PBMCs (from healthy or cancer patient donors) were stimulated with 0.5 μg / mL CMV peptide activator (Milteny Biotec, Cat. No. 130 - 093 - 435) with / without recombinant human CD28 (R&D system, Cat. No. 342 - CD), human CTLA-4 (R&D system, Cat. No. 434 - CT), human CD80 (R&D system, Cat. No. 140 - B1) at specified concentrations at 37°C in a 96-well plate for 2 - 5 days. For the SEB setting, 1×10 5 PBMCs were cultured with 0.5 ng / mL staphylococcal enterotoxin B (SEB) (Sigma, Cat. No. S4881) in the presence of rec. human CD28 at a specified concentration for 72 hours. If specified, anti-PD1 or human IgG was added at a final concentration of 5 μg / mL.

[0265] Autologous monocyte CD3 MLR -0.5×10 6 T cells were mixed with 0.5×10 5 monocytes from the same CMV-reactive donor and stimulated with 0.5 μg / mL CMV peptide activator with / without treatments at specified concentrations at 37°C for 6 days.

[0266] Stimulation of monocytes with recombinant human CD28 -1.5×10 were seeded in a 24-well plate in RPMI medium (R&D system, Cat. No. 285 - IF) with 100 - 100 U / ml IFNγ in the presence of recombinant human CD28 at a specified concentration.6 Monocytes for 48 hours. Designated phenotypes of cell populations generated by FACS analysis of relevant markers (IDO, PD-L1, and PD-L2) and by testing the secretion of characteristic cytokines (IL-6).

[0267] T cell stimulation with OKT3 - Stimulate 0.1×10 isolated CD3 T cells (from healthy donors) with the specified amount of anti-CD3 clone OKT3 at 37°C for 48 - 72 hours. When stating, add recombinant human CD80-Fc (2 μg / mL, R&D system) in soluble form. Add antibodies or VHH to CD28 or control at the specified concentration in soluble form. 6 Isolated CD3 T cells (from healthy donors) for 48 - 72 hours. When stating, add recombinant human CD80-Fc (2 μg / mL, R&D system) in soluble form. Add antibodies or VHH to CD28 or control at the specified concentration in soluble form.

[0268] Co - culture of SCC - 25 cancer cell line with monocytes in a transwell - based assay - Plate 4×10 SCC-25 cells on the bottom of a 24-well plate with 1×10 monocytes placed on a cell culture insert (Millipore, Cat. No. MCHT241148), where the treatment is carried out or not in serum-free starvation medium for 4 days. 4 Isolated CD3 T cells (from healthy donors) for 48 - 72 hours. When stating, add recombinant human CD80-Fc (2 μg / mL, R&D system) in soluble form. Add antibodies or VHH to CD28 or control at the specified concentration in soluble form. 5 Isolated CD3 T cells (from healthy donors) for 48 - 72 hours. When stating, add recombinant human CD80-Fc (2 μg / mL, R&D system) in soluble form. Add antibodies or VHH to CD28 or control at the specified concentration in soluble form.

[0269] Detection of soluble human CD28 in the plasma of cancer patients - Purchased 20 frozen plasma samples for each of 10 different cancer indications and healthy donors from DxBiosamples (San Diego, CA, USA). Plasma samples were diluted 1:20 and analyzed for soluble human CD28 by ELISA. Samples with high CD28 were analyzed again at appropriate dilutions.

[0270] Direct CD28 EIA - Unless otherwise discussed, screening was performed using Corning high-binding plates or equivalent plates. Coat 200 - 300 ng of human CD28-Ig chimera (R&D, Cat. No. 342-CD), mouse CD28-Ig chimera (R&D, Cat. No. 483-CD), or a BSA-conjugated dimer peptide composed of the amino acid sequence of the CD28 stem region (Gly137 - Pro152) per well. Block the plates with 5% milk or 1% casein in PBS for 1 hour at room temperature (RT). Wash the plates 3 times with PBST and incubate with the research antibody after detection with goat anti-mouse HRP Fc diluted 1:5000 specifically. The positive control was mouse anti-human CD28 clone 28.2 or mouse serum from immunized mice. Un-diluted screened hybridoma supernatant cultures.

[0271] Antibody sequencing. The antibody was provided to Rapid Novor for amino acid sequencing. Sequencing was performed using standard methods, which briefly included LC-MS analysis after enzymatic digestion with six enzymes (pepsin, trypsin, chymotrypsin, elastase, Lys C, and AspN). Digestion was performed with disulfide reduction and alkylation. LC-MS / MS analysis was performed using a Thermo-Fisher Q-exactive mass spectrometer. In the heavy and light chains of each antibody, at least 5 peptide scans covered 100% of the amino acid residues with significant supporting fragment ions. CDRs were determined using the Chothia scheme.

[0272] Example 1: Human CD28 undergoes proteolytic shedding during chronic stimulation

[0273] Soluble CD28 (sCD28) was detected by ELISA in cultures of chronically stimulated human PBMCs ( Figure 1 , upper panel). This phenomenon was evident regardless of the nature of the stimulant, either artificial (SEB) or physiological (CMV), indicating the robustness of the phenomenon. Treatment with TAPI-1 and GM6001 (broad MMP and ADAM17 inhibitors) reduced the amount of sCD28 in a dose-dependent manner, so the source of soluble CD28 is shedding from the membrane form ( Figure 1 , upper panel). Figure 2 As seen in Figure 2 , the cellular source of shed CD28 is T cells. Chronic stimulation of the Jurkat T cell line or human CD4 T cells from the peripheral blood of healthy donors with PHA resulted in the production of sCD28 in a dose-dependent manner ( Figure 2 , upper panel). Treatment with TAPI-1 and GM6001 reduced the amount of sCD28 in a dose-dependent manner at each PHA concentration ( Figure 2 , upper panel) and at a fixed PHA concentration (

[0274] , lower panel). Figure 3A Treatment with GI254023X (a highly specific ADAM-10 inhibitor) resulted in almost complete inhibition of the release of sCD28 from activated immune cells and in a dose-dependent manner ( Figure 3B , lower panel). Similar results were observed using the ADAM-17 specific inhibitor TMI-1 ( Figure 3A -B, upper panel). The viability of immune cells was monitored by checking the metabolic activity of cells in the cultures by MTT assay. The results showed that there was no significant difference between treatments with and without any of the ADAM inhibitors, meaning that the low sCD28 levels were caused by blocking protease activity rather than being an artifact of cell death caused by protease inhibitors (

[0275] The production of sCD28 has also been verified in more physiological systems. First, isolated autologous dendritic cells and CD4 T cells that mimic the physiological stimulation of T cells by antigen-presenting cells were used. When the two cell populations were mixed, the elevation of sCD28 was evident and became more prominent when CMV was added to the culture ( Figure 4A ). This indicates that when chronic stimulation occurs, the human CD28 protein undergoes a proteolytic shedding process.

[0276] Next, human PBMCs were stimulated with CMV peptides ( Figure 4B ) or SEB ( Figure 4C ) for 24 hours. Then, the cells were washed to remove the stimulant and replated without any stimulation for various time periods. Subsequently, the presence of sCD28 in the culture medium was examined. Over time, the accumulation of sCD28 was clearly visible. In addition, the accumulation depends on the activities of ADAM-10 and ADAM-17, as Figure 4D shows. Adding different concentrations of specific inhibitors led to a decrease in the amount of sCD28 quantified after 120 hours following SEB stimulation. This study can explain the presence of high amounts of soluble CCD28 in the blood of patients, since CD28 shedding occurs upon primary activation of T cells and does not necessarily require continuous or repeated stimulation.

[0277] Example 2: Soluble human CD28 has immunosuppressive activity

[0278] As Figure 1 (the figure below) shows, reducing the level of sCD28 using a protease inhibitor mixture is directly correlated with an increase in T cell activation, as shown by the level of secreted IFNγ, indicating that sCD28 has an immunosuppressive function. Increasing the concentration of the protease inhibitor mixture results in lower levels of sCD28 in the cell culture medium, and these lower levels of sCD28 are negatively correlated with higher levels of secreted IFMγ. To further explore the immunosuppressive effect of sCD28, recombinant human CD28 lacking the transmembrane and cytoplasmic domains was added to CMV-stimulated human PBMC cultures. This led to a dose-dependent inhibition of IFNγ secretion ( Figure 5 ). This immunosuppressive effect was observed in different human PBMC donors, confirming the robustness of this signaling axis blocked by sCD28.

[0279] Meanwhile, interleukin-6 secretion ( Figure 6 and 7A ) and interleukin-10 ( Figure 7A) elevation is significant. It has been reported that these cytokines exhibit inhibition of immune effector activity (IL-10) and skew the immune system towards a type 2 immune response, which can support cancer proliferation and angiogenesis through STAT-3 signaling (IL-6). In addition, a comparison was made with soluble CTLA-4 (mimicking abatacept - a registered therapy for autoimmune disorders), and it was revealed that there is an overall similar effect on the immune system in terms of cytokine secretion profiles ( Figure 7A ).

[0280] Next, human PBMCs were stimulated with SEB (1 ng / mL) in the absence or presence of recombinant human CD28. Human IgG was used as a control. Lymphocyte aggregation was monitored using S3Live-Cell to mark immune activation, and pictures were taken every 12 hours. As Figure 7C can be seen, SEB had essentially no effect on lymphocytes in the presence of recombinant human sCD28. It is well known that during an in vitro immune response, antigen-presenting cells (APCs) aggregate with each other and with other cell types, and aggregation is essential for the antigen-specific activation of resting lymphocytes. During the SEB immune response, soluble CD28 seems to reduce the amount and size of aggregate formation, meaning it inhibits the first step of APC-mediated T cell-specific activation.

[0281] Similar results were observed when isolated autologous monocytes and CD3 T cells were co-cultured in a mixed lymphocyte reaction (MLR). The mixed cells were stimulated with CMV peptide (0.5 μg / mL) for 5 days in the presence or absence of increasing concentrations of recombinant human sCD28. Again, sCD28 was found to inhibit IFNγ secretion while increasing the secretion of IL-1B, TGFβ, and IL-10 ( Figure 7B ).

[0282] sCD28 has a similar immunosuppressive effect on monocytes. Indoleamine 2,3-dioxygenase (IDO) has been involved in immune regulation through its ability to catabolize the essential amino acid tryptophan. It is expressed by different immune cells and also by many cancer cells. Tryptophan deficiency inhibits the maturation and proliferation of T lymphocytes, while kynurenine, the end product of tryptophan catabolism, is also known as an immunosuppressive metabolite that promotes immune tolerance in various physiological and pathological conditions. To test the effect of sCD28 on IDO, isolated human monocytes were stimulated with IFNγ (1000 U / mL) for 48 hours in the presence of control human IgG or recombinant human CD28 (10 μg / mL). After incubation, the monocytes were stained intracellularly for human IDO ( Figure 7E)。To promote intracellular staining, cells were fixed and permeabilized using the BD Cytofix / Cytoperm Buffer Kit. The IDO activity of the media from different treatments was evaluated using the ImmuSmol specific kynurenine ELISA kit( Figure 7D )。sCD28 strongly enhanced IDO expression in monocytes.

[0283] Furthermore, it was surprisingly found that sCD28 is an effective 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 the secretion of pro-inflammatory cytokines (IFNγ and IL-2), but the presence of sCD28 completely abolished this immune activation( Figure 8A )。

[0284] Similar results were again observed in the MLR setting. The MLR was run as before only in the presence and absence of sCD28 and in the presence and absence of the 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.

[0285] To elucidate the mechanism by which sCD28 inhibits the pro-activation effect of anti-PD-1 therapy, the expression of PD-1 ligands on immune cells was examined in the presence of sCD28. Isolated human monocytes were stimulated with IFNγ (1000 U / mL) for 48 hours in the presence of control human IgG (10 μg / mL) or recombinant human CD28 (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 a possible way by which sCD28 may circumvent the effects of anti-PD-1 immunotherapy.

[0286] Example 3: Soluble human CD28 was found in the plasma of cancer patients

[0287] The levels of sCD28 in cancer have been shown only in a few breast cancer patients and were found to be only slightly higher than those observed in healthy individuals ((Isitmangil, G., In vivo, 2016). Although the authors suggested that sCD28 could be used as a biomarker for breast cancer, no functional relationship was proposed. It is now known that soluble CD28 may actually enhance cancer immune escape, and 220 samples covering 10 different cancer indications and 20 samples from healthy donors were investigated. High sCD28 levels were found in several cancers, sometimes several orders of magnitude higher than those seen in healthy controls or even breast cancer patients ( Figure 9A ). In fact, when compared to the sCD28 levels found in some melanoma, rectal cancer, non-small cell lung cancer, and head and neck cancer patients, the levels in breast cancer patients seem comparable to those in healthy individuals.

[0288] To further clarify the role of sCD28 in cancer, PBMCs were isolated from cancer patients with different indications. Cells were stimulated for 3 days with SEB (5 ng / mL) alone, or in combination with MK-3475, recombinant human sCD28, or a combination of the two molecules. In the presence of sCD28, the concentration of human IFNγ in the cell supernatants from all donors was greatly reduced, even in the presence of MK-3475 ( Figure 9B ). In fact, sCD28 abolished the effect of MK-3475.

[0289] Next, cells of the head and neck cancer cell line SCC-25 were incubated alone or with monocytes in a transwell assay. SCC-25 cells grown alone were treated with IL-6 as a positive control, and indeed cell proliferation increased, as measured by MTT ( Figure 9C , upper) and by % confluence ( Figure 9C , lower). Cancer cell growth in the presence of monocytes also increased proliferation, but the greatest increase observed so far was co-culture including sCD28. This data further supports that sCD28 has a pro-cancer effect.

[0290] Example 4: sCD28 inhibits the efficacy of CD80-Fc

[0291] CD80 is one of the two major ligands of mCD28 together with CD86. The extracellular domain of CD80 fused to the Fc portion has been used as an immune-stimulatory molecule and is being investigated as a cancer therapy. To examine the effect of CD28 on the efficacy of CD80-Fc, isolated human CD3 T cells were stimulated with plate-bound anti-CD3 antibody (OKT3, 2 μg / mL) in the presence of 2 μg / mL soluble recombinant human CD80-Fc. As expected, CD80-Fc increased IFNγ secretion. However, the addition of sCD28 abrogated the secondary activation effect of CD80-Fc ( Figure 10A ). Similarly, when isolated PBMCs were stimulated with CMV peptides for 3 days and then incubated with sCD28, an increased amount of CD80-Fc was required to generate the expected immune response ( Figure 10B ).

[0292] Example 5: Effect of sCD28 on cancer in vivo

[0293] Since mice do not cleave mCD28, the role of sCD28 cannot be easily examined in a mouse model. The closest option is to administer recombinant sCD28 to mice to mimic elevated sCD28 levels. This was investigated in an H22 syngeneic mouse model. Balb / c fully immunocompetent mice received an allograft of H22 hepatocellular carcinoma cells. The cells grew even in fully immunocompetent mice, while the addition of anti-PD-1 therapy almost completely abolished tumor growth ( Figure 11A ). When recombinant human sCD28 was added, the effect of anti-PD-1 therapy almost completely disappeared in two mice ( Figure 11B ). This indicates that in some subjects, an increase in sCD28 levels can have a highly detrimental effect on cancer progression.

[0294] Example 6: Characterization of anti-shedding antibody-based reagents

[0295] The discovery that human CD28 undergoes proteolysis by ADAM10 and ADAM17 prompted an examination of candidate regions of its polypeptide sequence, showing potential susceptibility to proteolytic shedding. Studies have shown that ADAM10 and ADAM17 prefer leucine, valine, and aromatic residues at the P1' site. The most intriguing sequence region in human CD28 is the stalk segment, ranging 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 3 leucine and valine residues, as well as a phenylalanine residue, and is not expected to have any secondary structure elements that might impede protease access. Notably, the stalk region also contains cysteine 141, which forms an intermolecular disulfide bond that promotes the homodimerization of CD28. To generate antibodies or antibody fragments that specifically bind to the CD28 stalk region and may block access of different proteases to shed CD28, while avoiding any impairment of the CD28 oligomeric structure and function, CD1 mice were immunized with a dimeric peptide mimicking the CD28 stalk region. The peptide sequence used for immunization was SEQ ID NO:29, GKHLCPSPLFPGPSKPK, with a C-terminal lysine added to have a free amino group to allow conjugation to KLH or BSA using hydrazide chemistry. 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.

[0296] Antibodies with high binding affinity for recombinant human CD28 were discovered by direct CD28 EIA measurement. The antibody was named M9 and the sequence of the antibody was provided above. Serial dilutions of antibody M9 were used to confirm its specific binding to recombinant human sCD28 and the stalk region peptide ( Figure 12A ). Interestingly, while the antibody was able to detect recombinant human sCD28, it was unable to detect sCD28 actually shed from immune cells ( Figure 12B ). This strongly suggests that the antibody binds at the cleavage site and that the epitope it binds is incomplete in the cleaved form.

[0297] Next, the ability of the antibody to bind mCD28 on the cell surface was investigated. To reduce the shedding of sCD28 from cells, the antibody needed to bind to the membrane form of the protein and not just the recombinant protein in solution. HEK293 cells overexpressing human full-length CD28 were analyzed. Mouse CD28 did not appear to be cleaved into a soluble form (activated mouse splenocytes did not appear to produce sCD28), and thus the human protein had to be studied. Cells were analyzed by flow cytometry using the M9 antibody and the CD28.2 antibody as a positive control. Surprisingly, M9 did not appear to bind to surface mCD28 ( Figure 12C) This may be due to steric hindrance and limited access to the stem region when it is adjacent to the membrane.

[0298] Example 7: Inhibition of shedding of sCD28 from the cell surface by a single domain antibody

[0299] Small reagents were designed that could bind to mCD28 on the cell surface and block the shedding of sCD28. While the size of a full-size antibody is approximately 150 kDa, the Fab fragment derived from an antibody has a size of approximately 50 kDa, the single-chain antibody (also known as single-chain variable fragment, scFvs) has a size of approximately 25 kDa, and the single domain antibody (also known as VHH antibody, scFvs, and DARPins) has a size of only 12 - 15 kDa.

[0300] Isolation of single domain antibodies using a phage library of llama-derived VHHs used for the first time in experiments. The library consists of VHH sequences taken from non-immunized llamas used for the first time in experiments, i.e., B cells were extracted and the entire available repertoire of VHH CDRs was sequenced. These CDRs were introduced into phages to generate the library. Using ELISA and flow cytometry, the library was screened against the recombinant CD28 extracellular domain and the dimer stem region to discover antibodies that specifically bind to the stem region of human C28. The VHH sequences that specifically bind to the stem region of human CD28 were: EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVT VSSAAAHHHHHH (SEQ ID NO:45, clone 2A1); EVQLVESGGGLVQAGGSLRLSCAASGSLFSINAMAWYRQAPGKQRELVAAITSSGSTNYANSVKGRFTVSRDNAKNTMYLQMNSLKPEDTAVYYCVVDEYGSDYWIWGQGTQVTVSSAAAHHHHHH (SEQ ID NO:46, clone 4A4); and QVQLVESGGGLVQAGGSLRLSCAASGSIFSINAMGWYRQAPGKQRERVAAITSGGSTNYADSVKGRFTISRDNAKNTVYLQMNNLEPRDAGVYYCVVDLYGEDYWIWGQGTQVTVSSAAAHHHHHH (SEQ ID NO:47, clone 4A1). The VHHs were produced as recombinant proteins in CHO cells and then cell binding and anti-shedding activity were evaluated as described below. The C-terminal His-tag was used for purification and linked by a triple alanine repeat. The CDRs of the three studied clones are provided in Table 1.

[0301] Table 1

[0302] VHH clones CDR1 (SEQ ID) CDR2 (SEQ ID) CDR3 (SEQ ID) 2A1 INAMG(33) AISGGGDTYYADSVKG(34) DLYGSDYWD(35) 4A4 INAMA(36) AITSSGSTNYANSVKG(37) DEYGSDYWI(38) 4A1 INAMG(33) AITSGGSTNYADSVKG(39) DLYGEDYWI(40)

[0303] Binding of VHH clones to the human CD28 stem region sequence was first confirmed by ELISA using serial dilutions of the VHH clones ( Figure 13 ). Binding of membrane human CD28 at the cell level was confirmed by FACS analysis using labeled VHH clones and HEK cells overexpressing CD28 ( Figure 14)。Membrane CD28 binding demonstrated entry into the CD28 membrane-proximal region. Previous experiments have shown that the size of the reagent is crucial for entry into this region, as full-size antibodies that can bind to the CD28 stalk region peptides cannot bind to the CD28 stalk region on cells. Notably, the VHH clones cannot bind to the human CD28 stalk region sequence with an L-K substitution at amino acid residue 145 within the MMP cleavage site( Figure 23 )。

[0304] Anti-shedding activity was confirmed both at the peptide and cellular levels. ELISA technology was used to detect intact human CD28 stalk region dimer peptides to confirm that the VHH clones blocked MMP-2( Figure 17 ) and MMP-13( Figure 22 ) cleavage of the human CD28 stalk region. Although the M9 Fab exhibited the ability to block MMP-2 cleavage of the CD28 stalk region peptide, as described above it could not bind to the CD28 stalk region on cells and could not inhibit CD28 shedding on the cell membrane. At the cellular level, a standard sandwich ELISA was used to confirm the efficacy of the VHH clones in inhibiting sCD28 shedding by measuring the levels of human sCD28 in the supernatants of HEK cells overexpressing human CD28( Figure 18 ), PHA- and IL-2-activated isolated CD4 T cells( Figure 19 ) and superantigen-activated PBMCs( Figure 20 ). As expected, the M9 Fab did not reduce the sCD28 levels in the supernatants, further highlighting the importance of the size and architecture of the blocker for its actual ability to block shedding.

[0305] Importantly, it was found that the VHH clones did not impair human CD28 functionality. Using flow cytometry, it was found that the VHH clones did not alter the magnitude of CD86 binding to membrane CD28( Figure 15 .). A standard sandwich ELISA was used to show that the VHH clones did not agonize CD28, as measured by the secretion level of the inflammatory cytokine interferon γ( Figure 16 ). The activating antibody CD28.2 was used as a positive control. Similarly, a standard sandwich ELISA was used to show that the VHH clones did not antagonize CD80-Fc stimulation through CD28, as measured by the secretion level of the cytokine IL-2( Figure 21 ).

[0306] Example 8: Design of other small reagents to inhibit sCD28 shedding on the cell surface

[0307] Fab fragment generation was performed using a commercial kit or commercial service. The CDR regions of antibody M9 were used for Fab generation as they have been shown to bind to the appropriate deisotope. The efficacy of the resulting Fab fragments was first tested by binding assays with recombinant human CD28 and the dimer stem region peptide to confirm that this binding was retained. Binding to surface mCD28 of murine cells expressing human CD28 and human immune cells was assayed by FACS. Antibody CD28.2 was used as a positive control. Direct inhibition of sCD28 shedding was tested in immune cell cultures after stimulation. sCD28 in the culture medium was measured by sandwich ELISA in the presence and absence of Fab fragments. The effect of Fab fragments with shedding inhibitory activity on CD28 signaling was determined. First, agonistic activity was tested by determining the ability of the Fab fragments to induce the secretion of pro-inflammatory cytokines, such as interferon γ, from T cells. Second, the ability to block CD80-Fc (agonist) binding was used to test the anti-antagonistic properties of the Fab fragments.

[0308] Single-chain antibody generation using the M9 CDRs was performed by standard methods, using a commercial service, or by inserting the CDRs into an scFV scaffold. Purification was performed and the resulting antibodies were evaluated by the same assays as for the Fab fragments.

[0309] Single-domain antibodies were generated by one of two strategies. 1) Library first used for experiments - a phage library of VHHs from llamas first used for experiments - the library consisted of VHH sequences taken from the spleen of a llama first used for experiments, i.e., B cells were extracted and the entire available library of VHH CDRs was sequenced. These CDRs were introduced into phage to generate a library. The library was screened against the recombinant CD28 extracellular domain and the dimer stem region to discover antibodies that specifically bind sCD28. 2) Immunized library - llamas or other animal families or sharks were immunized with cells overexpressing CD28. After cellular immunization, the spleen was extracted and the available library of VHH CDRs was sequenced. The extracted splenic B cells were made into hybridomas. The resulting antibodies were introduced into phage to generate a library and the library was screened against the recombinant CD28 extracellular domain and the dimer stem region peptide to find antibodies that specifically bind sCD28. The shedding-blocking and agonistic / antagonistic activities of single-domain antibodies with specific binding were evaluated as for the Fab fragments and single-chain antibodies.

[0310] Although the invention has been described in connection with its specific embodiments, it is apparent 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

1. A method for generating a single-domain antibody (sdAb) that inhibits proteolytic cleavage of membrane CD28 (mCD28) on the cell surface, comprising at least one of the following: a. Obtaining an sdAb that binds to the extracellular domain of CD28 or a fragment thereof; b. Testing the ability of the obtained sdAb to block proteolytic cleavage of the CD28 stalk domain by a protease; c. Selecting an sdAb that blocks proteolytic cleavage of the CD28 stalk domain by a protease; d. Testing the binding of the selected sdAb to mCD28 on the cell surface; e. Selecting an sdAb that binds to cell surface mCD28; and f. Culturing a host cell comprising one or more vectors, the one or more vectors comprising a nucleic acid sequence encoding the sdAb, wherein the nucleic acid sequence is the nucleic acid sequence of an sdAb selected by: i. Obtaining an sdAb that binds to the extracellular domain of CD28 or a fragment thereof; ii. Testing the ability of the obtained sdAb to block proteolytic cleavage of the CD28 stalk domain by a protease; iii. Selecting an sdAb that blocks proteolytic cleavage of the CD28 stalk domain by a protease; iv. Testing the binding of the selected sdAb to mCD28 on the cell surface; and v. Selecting an sdAb that binds to cell surface mCD28; Thereby generating an sdAb that inhibits proteolytic cleavage of mCD28 on the cell surface.

2. The method according to claim 1, wherein the CD28 stalk domain comprises the amino acid sequence GKHLCPSPLFPGPSKP (SEQ ID NO:9) or KGKHLCPSPLFPGPS (SEQ ID NO:27).

3. The method according to claim 1 or 2, wherein the protease is selected from MMP-2 and MMP-13.

4. The method according to any one of claims 1 to 3, wherein the sdAb that binds to the extracellular domain of CD28 or a fragment thereof is obtained by a method comprising at least one of the following steps: a. Immunizing a shark or camelid with the extracellular domain of CD28 or a fragment thereof and collecting antibodies from the immunized organism; and b. Screening a library of reagents that bind to the extracellular domain of CD28 or a fragment thereof and selecting a bound reagent.

5. The method according to claim 4, wherein the extracellular domain of CD28 or a fragment thereof is dimeric or monomeric.

6. The method according to claim 4 or 5, wherein collecting the antibodies comprises extracting B cells from the spleen of the immunized shark or camelid.

7. The method according to any one of claims 4 to 6, wherein selecting the bound sdAb comprises sequencing the selected sdAb and generating a recombinant form of the sdAb from the sequence.

8. The method according to any one of claims 1 to 7, wherein the CD28 stalk domain is dimeric.

9. The method according to any one of claims 1 to 8, further comprising assaying mCD28 downstream signaling in the presence of the obtained sdAb and selecting at least one sdAb that neither substantially agonizes nor substantially antagonizes mCD28 signaling.

10. The method according to any one of claims 1 to 9, wherein the CD28 extracellular domain or fragment thereof is the CD28 stalk domain and comprises the amino acid sequence GKHLCPSPLFPGPKP (SEQ ID NO:9) or KGKHLCPSPLFPGPS (SEQ ID ID:27).

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