Methods and compositions for reducing soluble immunoreceptor CD28

Abstract

The present application relates to methods and compositions for reducing soluble immune receptor CD28. Methods of treating cancer and improving immunotherapy are provided, including reducing soluble immune receptor levels. Also provided are agents that bind to membrane immune receptors and inhibit protease cleavage of the receptors and agents that bind to soluble immune receptors and are neither receptor agonists nor antagonists, as well as methods of producing these agents.

Description

[0001] This application is a divisional application. The application date of the original application is March 14, 2019, the application number is 201980029648.2, and the name of the invention is “Methods and compositions for reducing soluble immune receptor CD28”, which is incorporated herein by reference in its entirety.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 643,334, filed on March 15, 2018, U.S. Provisional Patent Application No. 62 / 643,355, filed on March 15, 2018, and U.S. Provisional Patent Application No. 62 / 774,254, filed on December 2, 2018, which are incorporated herein by reference in their entireties. Technical Field

[0004] The invention belongs to the field of immunomodulation and immunotherapy. Background Art

[0005] The adaptive immune system plays a key role in regulating and defending against pathogens and cancer cells, primarily through the coordinated stimulation of antigen-specific cofactor CD4+ and cytotoxic CD8+ T cells. The persistent and sustained activation of T cells by antigen presenting cells (APCs) involves: i) the interaction (engagement) of the T cell receptor (TCR) with peptides presented by the major histocompatibility complex (MHC) on the APC; ii) the binding of the co-stimulatory CD28 receptor on the T cell to the B7-1 (CD80) and B7-2 (CD86) ligands also expressed by the APC. The biological consequences of CD28 co-stimulation are diverse and include control of the T cell cycle, expansion, differentiation, and amplification of TCR stimulation by lowering the threshold required to achieve immune effector functions.

[0006] In contrast to 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 triggering by CD28. Both CTLA-4 and CD28 are type I transmembrane proteins. Their extracellular parts consist of a V-set immunoglobulin superfamily (Ig-V) domain, which is homo-covalently linked by cysteine ​​residues located outside the IgV domain, adjacent to the transmembrane region. Despite their similarities, CTLA-4 and CD28 differ in affinity and quaternary structural arrangement. CTLA-4 was found to have a higher binding affinity for B7 molecules and a different dimerization mode than CD28, resulting in different stoichiometric binding to the shared ligand. CD28 exhibits monovalent binding stoichiometry, while CTLA-4 interacts in a bivalent manner. Thus, CTLA-4 binds to B7 molecules with much higher affinity and avidity than CD28, thus downregulating T cell responses and favoring the initiation of antigen-specific tolerance.

[0007] It has been shown that some co-stimulatory molecules have several physiological forms. In addition to the membrane-bound form, a soluble form expressed in immature immune cells has also been described, which increases the complexity of T cell biology. The soluble form of CD28 (sCD28) has been attributed to the gene product of alternative splicing. The splicing event causes frameshifting, with the result that two glutamic acid residues are added after the glycine at position 137 before translation termination. The final product lacks the entire transmembrane and cytoplasmic region, and importantly lacks the cysteine ​​residues (Magistrelli G., Biochem Biophy Res Commun, 1999) that mediates the disulfide bond of dimer CD28 at position 141. The biological function and anti-receptor binding of the monomeric CD28 soluble form are examined (Hebbar, M., Clin Exp Immunol, 2004) and it is shown that T cell proliferation is also inhibited. However, in the case of dimeric sCD28, it has been shown to have a regulatory effect of inhibiting T cell functionality by binding to B7 molecules (Sun, Z., Centr Eur J Immunol, 2014; Hebbar, M., Clin Exp Immunol, 2004). It is worth noting that an increase in the number of sCD28 molecules in the serum of patients with autoimmune disorders has been reported (Wong, CK, Rheumatol, 2005; Hamzaoui, K., Clin Exp Rheumatol, 2005; Hebbar, M., Clin Exp Immunol, 2004; Sun, Z., Clin Immunol, 2014). The exact origin of sCD28 is still controversial. Using an in vitro model of T cell activation, reflecting the persistent inflammatory state of T cells in autoimmune patients, it has been shown that during T cell activation, transcription of the alternative soluble form is suppressed and only the full-length membrane form of CD28 is evident, while the amount of sCD28 in culture is elevated (Hebbar, M., Clin Exp Immunol, 2004). This phenomenon has led to the claim that active shedding of the membrane form of CD28 is responsible for the elevation of the soluble molecule in serum, however, this has not been confirmed. Active shedding during T cell activation has been described in the past as a regulatory mechanism that counteracts continued activation by proteolysis of adhesion molecules.

[0008] While CTLA-4 limits the magnitude of early T cell responses, another inhibitory receptor, PD-1, inhibits peripheral T cell function. PD-1 expression increases during T cell activation, and its known ligands are homologs of the B7 family: B7-H1 (PD-L1) and B7-H2 (PD-L2). These homologs have been found on APCs and cancer cells and drive activated T cells into a state of cellular anergy, resulting in a weakened immune response. Therefore, targeted therapies involving the CTLA-4 and PD-1 / PD-L1 axes have shown clinical activity in a wide variety of cancer types. Recently, studies have shown that the CD28 signaling pathway is targeted and inhibited by PD-1 (Hui, E., Science, 2017), while a complete active CD28 / B7 axis is essential for effective PD-1 therapy (Kamphorst, AO, Science, 2017).

[0009] However, not all patients respond to PD-1-based immunotherapy or immunotherapy in general, and those who do often relapse. Therefore, methods and molecules that can improve the ability of patients' immune cells to attack cancer are urgently needed. Summary of the invention

[0010] The present invention provides a method for treating cancer and improving PD-1 / PD-L1-based immunotherapy, the method comprising reducing the level of soluble CD28. Also provided are agents that bind to membrane CD28 and inhibit mCD28 proteolytic cleavage and agents that bind to soluble CD28 and are neither CD28 agonists nor CD28 antagonists, as well as methods for producing these agents.

[0011] According to a first aspect, there is provided a method of treating and / or preventing cancer in a subject in need thereof, the method comprising reducing the level of soluble CD28 (sCD28) in the subject.

[0012] According to another aspect, a method of improving PD-1 and / or PD-L1 based immunotherapy in a subject in need thereof is provided, the method comprising reducing sCD28 levels in the subject.

[0013] According to another aspect, an agent is provided that binds soluble CD28 (sCD28) and is neither a CD28 agonist nor a CD28 antagonist.

[0014] According to some embodiments, the subject in need of immunotherapy has cancer. According to some embodiments, the subject is unresponsive or poorly responsive to immunotherapy based on PD-1 and / or PD-L1.

[0015] According to some embodiments, the reduction occurs in the blood, peripheral blood, or tumor microenvironment of the subject. According to some embodiments, the reduction comprises at least one of:

[0016] a. administering to a subject an agent that binds to sCD28, and wherein the agent degrades sCD28 upon binding or targets sCD28 for degradation;

[0017] b. administering to a subject an inhibitory nucleic acid molecule, wherein the molecule binds to mRNA encoding sCD28 but not to mRNA encoding membrane CD28 (mCD28);

[0018] c. administering to a subject an agent that binds to mCD28 and inhibits protease cleavage of mCD28;

[0019] d. administering to a subject a dimeric peptide comprising the stalk region of human CD28;

[0020] e. administering to the subject an agent that inhibits a protease capable of cleaving mCD28, and

[0021] f. Drawing blood from the subject, reducing the amount of sCD28 in the blood and returning the blood to the subject.

[0022] According to some embodiments, the stem region,

[0023] a. comprising the amino acid sequence GKHLCPSPLFPGPSKP (SEQ ID NO: 9) or KGKHLCPSPLFPGPS (SEQ ID NO: 36);

[0024] b. consisting of the amino acid sequence HVKGKHLCPSPLFPGPSKP (SEQ ID NO: 10); or

[0025] c. Both (a) and (b).

[0026] According to some embodiments, the method does not degrade mCD28 or does not reduce mCD28-mediated immune cell activation.

[0027] According to some embodiments, prior to the lowering, the subject's blood comprises at least 5 ng / ml sCD28.

[0028] 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. According to some embodiments, the cancer is selected from melanoma, head and neck cancer, non-small cell lung cancer, ovarian cancer and colorectal cancer.

[0029] According to some embodiments, the method of the present invention further comprises administering to the subject another immunotherapy. According to some embodiments, the immunotherapy is selected from:

[0030] a. Checkpoint inhibitors;

[0031] b. Chimeric Antigen Receptor (CAR)-based therapies; and

[0032] c.Cancer vaccines.

[0033] According to some embodiments, the checkpoint inhibitor is a PD-1 and / or PD-L1 based immunotherapy.

[0034] According to another aspect, an agent is provided that binds membrane CD28 (mCD28) and inhibits proteolytic cleavage of mCD28.

[0035] According to some embodiments, the agent is neither a CD28 agonist nor a CD28 antagonist. According to some embodiments, the agent neither degrades mCD28 nor inhibits mCD28-mediated immune cell activation.

[0036] According to some embodiments, the reagent

[0037] a. Does not induce antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC);

[0038] b. Contains IgG2 or IgG4;

[0039] c. comprising an Fc domain engineered to reduce CDC, ADCC, or both;

[0040] d. lack of Fc domain;

[0041] e. is a Fab fragment;

[0042] f. is a single-chain antibody;

[0043] g. is a single domain antibody;

[0044] h. is a small molecule;

[0045] i. a peptide that specifically binds to mCD28; or

[0046] j. Combinations thereof.

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

[0048] a. comprising the amino acid sequence GKHLCPSPLFPGPSKP (SEQ ID NO: 9) or KGKHLCPSPLFPGPS (SEQ ID NO: 36);

[0049] b. consisting of the amino acid sequence HVKGKHLCPSPLFPGPSKP (SEQ ID NO: 10); or

[0050] c. Both (a) and (b).

[0051] According to some embodiments, the agent inhibits proteolytic cleavage of at least one protease. According to some embodiments, the at least one protease is at least one metalloprotease. According to some embodiments, the at least one metalloprotease is selected from ADAM 10 and ADAM 17.

[0052] According to some embodiments, the agent is an antibody or antigen-binding fragment thereof, and comprises three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein:

[0053] CDR-H1 comprises the amino acid sequence shown in SEQ ID NO:30 (GFTFSSYYMS), CDR-H2 comprises the amino acid sequence shown in SEQ ID NO:31 (TISDGGDNTYYAGTVTG), CDR-H3 comprises the amino acid sequence shown in SEQ ID NO:32 (IHWPYYFDS), CDR-L1 comprises the amino acid sequence shown in SEQ ID NO:33 (RASSSVSYMN), CDR-L2 comprises the amino acid sequence shown in SEQ ID NO:34 (ATSDLAS), and CDR-L3 comprises the amino acid sequence shown in SEQ ID NO:35 (QQWSSHPPT).

[0054] According to some embodiments, the reagent of the present invention comprises at least one of the following

[0055] a. a heavy chain comprising the amino acid sequence of SEQ ID NO: 53; and

[0056] b. a light chain comprising the amino acid sequence of SEQ ID NO:55.

[0057] According to some embodiments, the agent does not bind membrane CD28 (mCD28).

[0058] According to some embodiments, the agent is selected from an antibody or antigen-binding fragment thereof, a Fab fragment, a single chain antibody, a single domain antibody, a small molecule and a peptide that specifically binds to sCD28.

[0059] According to some embodiments, binding of an agent to sCD28 in an organism results in at least one of the following:

[0060] a. Degradation of bound sCD28;

[0061] b. Removal of sCD28 from the blood; and

[0062] c. transporting the bound sCD28 to lysosomes, endosomes, proteasomes, or a combination thereof.

[0063] According to some embodiments, the agent does not inhibit binding of sCD28 to a ligand. According to some embodiments, the agent binds to dimeric sCD28, monomeric sCD28, or both. According to some embodiments, the agent binds outside the IgV domain of sCD28.

[0064] According to some embodiments, the agent is an antibody or antigen-binding fragment thereof, and comprises three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein:

[0065] CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO:12 (GYTLTNY), CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO:13 (NTYTGK), CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO:14 (GDANQQFAY), CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO:15 (KASQDINSYLS), CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO:16 (RANRLVD), and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO:17 (LQYDEFPPT);

[0066] CDR-H1 comprises the amino acid sequence shown in SEQ ID NO:18 (GYTFTSY), CDR-H2 comprises the amino acid sequence shown in SEQ ID NO:19 (YPGDGD), CDR-H3 comprises the amino acid sequence shown in SEQ ID NO:20 (NYRYSSFGY), CDR-L1 comprises the amino acid sequence shown in SEQ ID NO:21 (KSSQSLLNSGNQKNYLT), CDR-L2 comprises the amino acid sequence shown in SEQ ID NO:22 (WASTRES), and CDR-L3 comprises the amino acid sequence shown in SEQ ID NO:23 (QSDYSYPLT); or

[0067] CDR-H1 comprises the amino acid sequence shown in SEQ ID NO:24 (GYTFTDY), CDR-H2 comprises the amino acid sequence shown in SEQ ID NO:25 (NPNYDS), CDR-H3 comprises the amino acid sequence shown in SEQ ID NO:26 (SSPYYDSNHFDY), CDR-L1 comprises the amino acid sequence shown in SEQ ID NO:27 (SARSSINYMH), CDR-L2 comprises the amino acid sequence shown in SEQ ID NO:28 (DTSKLAS), and CDR-L3 comprises the amino acid sequence shown in SEQ ID NO:29 (HQRNSYPFT).

[0068] According to some embodiments, the reagents of the present invention include at least one of the following

[0069] a. a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 41, 45 or 49; and

[0070] b. a light chain comprising an amino acid sequence selected from SEQ ID NO: 43, 47 or 51.

[0071] According to some embodiments, the antigen binding fragment is selected from a Fv, Fab, F(ab')2, scFV or scFV2 ​​fragment.

[0072] According to some embodiments, the agent is humanized.

[0073] According to some embodiments, the agent does not induce antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0074] According to another aspect, there is provided a method for producing an agent of the invention, the method comprising:

[0075] Obtaining an agent that binds to the extracellular domain of CD28 or a fragment thereof, testing the ability of the agent to block the cleavage of mCD28 by a protease, and selecting at least one agent that blocks the cleavage of mCD28 by a protease; or

[0076] Cultivating a host cell containing one or more vectors comprising a nucleic acid sequence encoding an agent, wherein the nucleic acid sequence is a nucleic acid sequence of an agent selected by:

[0077] i. obtaining a reagent that binds to the extracellular domain of CD28 or a fragment thereof;

[0078] ii. testing the ability of the reagent to block cleavage of mCD28 by a protease; and

[0079] iii. selecting at least one reagent that blocks the cleavage of mCD28 by a protease;

[0080] Thereby, the reagent of the present invention is produced.

[0081] According to another aspect, there is provided a method for producing an agent of the invention, the method comprising:

[0082] Obtaining an agent that binds to the CD28 extracellular domain or a fragment thereof, measuring mCD28 downstream signaling in the presence of the obtained agent, and selecting at least one agent that neither substantially agonizes nor substantially antagonizes mCD28 signaling; or

[0083] Cultivating a host cell containing one or more vectors comprising a nucleic acid sequence encoding an agent, wherein the nucleic acid sequence is a nucleic acid sequence of an agent selected by:

[0084] i. obtaining a reagent that binds to the extracellular domain of CD28 or a fragment thereof;

[0085] ii. measuring mCD28 downstream signaling in the presence of the obtained reagent; and

[0086] iii. selecting at least one agent that neither significantly agonizes nor significantly antagonizes mCD28 signaling;

[0087] Thereby, the reagent of the present invention is produced.

[0088] According to some embodiments, the protease is selected from ADAM 10 and ADAM 17.

[0089] According to some embodiments, the agent that obtains specific binding to the extracellular domain of CD28 or a fragment thereof is an agent that obtains specific binding to the stalk domain of CD28.

[0090] According to some embodiments, the method of the present invention further comprises measuring mCD28 downstream signaling in the presence of the obtained agent, and selecting at least one agent that neither substantially agonizes nor substantially antagonizes mCD28 signaling.

[0091] According to some embodiments, the method of the present invention further comprises at least one of the following:

[0092] a. testing the obtained reagents for binding to mCD28 and selecting at least one reagent that does not bind to mCD28; and

[0093] b. Testing the obtained reagents for binding to sCD28 from a cancer patient, and selecting at least one reagent that binds to sCD28 from a cancer patient.

[0094] According to some embodiments, obtaining the reagent comprises at least one of the following:

[0095] a. Immunizing an organism with the extracellular domain of CD28 or a fragment thereof, and collecting antibodies from the immunized organism;

[0096] b. Screening a library of agents that bind to the CD28 extracellular domain or a fragment thereof, and selecting a binding agent.

[0097] According to some embodiments, the CD28 extracellular domain or fragment thereof is dimeric or monomeric.

[0098] According to some embodiments, the organism is selected from the group consisting of rabbit, mouse, rat, shark, camelid, chicken, goat and bacteriophage.

[0099] According to some embodiments, collecting antibodies comprises:

[0100] a. extracting B cells from the spleen of the immunized organism;

[0101] b. fusing the extracted B cells with myeloma cells to produce hybridomas; and

[0102] c. Collecting antibodies from hybridomas.

[0103] According to some embodiments, selecting a bound agent comprises sequencing the selected agent and generating a recombinant form of the agent from the sequence.

[0104] According to another aspect, there is provided an agent produced by the method of the invention.

[0105] According to another aspect, there is provided a pharmaceutical composition comprising an agent of the invention and a pharmaceutically acceptable carrier, excipient or adjuvant.

[0106] According to another aspect, a method of treating and / or preventing cancer in a subject in need thereof or improving PD-1 and / or PD-L1 based immunotherapy in a subject in need thereof is provided, the method comprising administering to the subject a pharmaceutical composition of the present invention.

[0107] According to another aspect, a method of determining the suitability of a subject to be treated by the methods of the invention is provided, comprising obtaining a sample from the subject and determining the level of sCD28 in the sample, wherein sCD28 levels above 5 ng / ml indicate that the subject is suitable for the treatment methods of the invention.

[0108] According to another aspect, a kit is provided, comprising at least one agent of the present invention.

[0109] According to some embodiments, the kit of the present invention further comprises at least one of the following:

[0110] a. Anti-PD-1 and / or PD-L1 immunotherapy;

[0111] b. a label stating that the agent of the invention is used in combination with a PD-1 and / or PD-L1 based immunotherapy; and

[0112] c. A second detection molecule for detecting at least one agent according to any one of claims 15-37 and 49.

[0113] According to the detailed description given below, other embodiments of the present invention and all applicable scopes will become obvious. However, it should be understood that the detailed description and specific examples, although indicating the preferred embodiments of the present invention, are only given by way of illustration, because various changes and modifications within the spirit and scope of the present invention will be obvious to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0114] Figure 1 . Soluble CD28 is produced during stimulation of PBMCs and is counteracted by the addition of protease inhibitors (PIs) Bar graph of the amount of soluble CD28 in PBMC cultures stimulated with SEB (0.5 ng / mL, left) or CMV peptide (0.5 μg / mL, right) quantified by human CD28 ELISA. Protease inhibitor cocktails were added at the indicated concentrations. Overall health and effector activity were examined by secretion of interferon gamma (bottom panel).

[0115] Figure 2 . Soluble CD28 is produced during PHA stimulation of T cells and is blocked by the addition of protease inhibitors. remove Bar graphs of Jurkat cells (upper left) or isolated human CD4 T cells (upper right) stimulated with increasing concentrations of PHA (1-4 μg / mL, top panel) in the presence of a fixed concentration (2 μM) of a protease inhibitor cocktail. In another setting, the concentration of PHA was fixed to stimulate Jurkat T cells (1 μg / mL PHA, lower left) or human CD4 T cells (2 μg / mL PHA, lower right), and the concentration of the protease inhibitor cocktail was titrated (0.5-2 μM). The concentration of human CD28 in the supernatant was quantified using a standardized sandwich ELISA.

[0116] Figure 3A -B. Specific ADAM-10 and ADAM-17 inhibitors abolish activation of human PBMCs by soluble CD28 in SEB accumulation during the period without hampering its viability (3A-B) Bar graphs of human PBMCs stimulated with SEB (1 ng / mL) in the presence of various concentrations (0.01-1 μM) of (3A) an ADAM-10 specific inhibitor (GI254023X) and (3B) an ADAM-17 specific inhibitor (TMI-1). The survival of cells in the different treatments was assessed using the MTT assay (upper panel). The concentration of human CD28 in the supernatant was quantified using a standardized sandwich ELISA (lower panel).

[0117] Figure 4A -D. Soluble CD28 production during PBMC stimulation . (4A) Bar graph of immature dendritic cells mixed with CD3 T cells from the same donor at a 1:5 ratio in the absence (black bars) or presence (dark gray bars) of CMV peptide. Controls for each cell population alone or with CMV are represented by light gray bars. The concentration of human CD28 in the supernatant was quantified by standardized sandwich ELISA. (4B-D) Bar graphs of human PBMCs stimulated for 24 days with (4B) CMV or (4C) SEB or (4D) SEB in the presence of ADAM-10 and ADAM-17 inhibitors and then transferred to clean cultures. Figure 4D Measurements in were 120 h after cell transfer.

[0118] Figure 5 . CD28 soluble splice variant is downregulated in activated cells . Photographs of gels showing the coding sequence of human CD28 amplified by PCR in unstimulated PBMC (lane 4) and CD4 (lane 6), stimulated PBMC (lane 5) and CD4 cells (lane 7). Amplified fragments were size separated on a 1% agarose gel and visualized by ethidium bromide. GADPH cDNA was amplified as a control (bottom). Bands used for Sanger sequencing are marked by S1 (PHA-activated CD4—650 bp) and S2 (naive CD4—550 bp band).

[0119] Figure 6 . Soluble CD28 inhibits the secretion of effector cytokines Bar graph of human PBMCs stimulated with CMV (0.5 μg / mL) in the absence (black bars) or presence (grey bars) of indicated concentrations of recombinant human CD28. Naive samples without CMV stimulation ( The concentration of human IFNγ in the supernatant was quantified by standardized sandwich ELISA (Biolegend).

[0120] Figure 7 . Soluble CD28 increases IL-6 cytokine secretion Bar graph of human PBMCs stimulated with CMV (0.5 μg / mL) in the absence (black bars) or presence (grey bars) of indicated concentrations of recombinant human soluble CD28. Naive samples without CMV stimulation are represented by light grey bars. The concentration of human IL-6 in the supernatant was quantified by standardized sandwich ELISA (Biolegend).

[0121] Fig. 8A-E. (8A) Line graph of human PBMC stimulated with CMV (0.5 μg / mL) in the presence of indicated concentrations of recombinant human soluble CD28 (grey triangles) or recombinant human soluble CTLA-4 (black circles). The concentrations of human IL-6, IFNγ, and IL-4 in the supernatant were quantified using standardized sandwich ELISA (Biolegend). The concentrations of human IL-8, IL-12p (40), and IL-10 in the supernatant were quantified using a multiplex assay using the Magpix system (Millipore). (8B) Bar graph of cytokine secretion from autologous monocytes and CD3 MLR. Naive samples without CMV stimulation are represented by light grey bars. CMV controls alone or CMV controls with IgG are represented by black bars. Increased concentrations of sCD28 are represented by dark grey bars. (8C) Line graph of lymphocyte cluster formation in human PBMCs stimulated with SEB in the presence of recombinant human soluble CD28 (grey circles), or with SEB together with control IgG (grey triangles). (8D) Bar graph of IDO secretion into culture measured by kynurenine ELISA kit from monocytes treated with and without recombinant human sCD28. (8E) Scatter plot of intracellular FACS of IDO in monocytes treated with and without recombinant human sCD28.

[0122] Fig. 9A -C. Soluble CD28 impedes anti-PD1 therapy (9A) Bar graphs of human PBMCs stimulated for 3 days with SEB (200 ng / mL, left panel) or CMV peptide (0.5 μg / mL, right panel) in the presence of anti-PD1 (MK3475, 5 μg / g, black bars) or recombinant human soluble CD28 (2 and 10 μg / mL, gray bars) or a combination of both (dashed bars). (9B) Bar graphs of cytokine secretion in the context of monocyte MLR, naive—white bars, CMV alone—light gray bars, sCD28—black bars, MK-3475—dark gray bars, sCD28+MK-3475—checkered bars. Concentrations of human IFNγ, TGFβ, and IL-2 in supernatants were quantified using standardized sandwich ELISA (Biolegend). (9C) Histograms of surface PD-L1 (left) and PD-L2 (right) expression in monocytes after incubation with control and sCD28.

[0123] Fig. 10A -D. Soluble CD28 in cancer patients. (10A) Dot plots show 20 plasma samples from each of the 10 cancer indications and healthy donors investigated for the presence of soluble human CD28. Samples high in soluble CD28 were repeatedly checked with several dilution factors. The concentration of human CD28 in the supernatant was quantified by a standardized sandwich ELISA with internal calibration to accommodate the readings from human plasma samples. (10B) Bar graph of ELISA detection of sCD28 from 20 melanoma patient samples using antibodies #1 and #3 of the present invention and a commercially available CD28 ELISA kit*—measured amounts exceeding 150 ng / ml. (10C) Bar graph of IFNγ secretion measured by sandwich ELISA from SEB-stimulated PBMCs of cancer patients (sarcoma patient—top left, renal cancer patient—top right, and two different head and neck cancer patients—bottom) in the presence of sCD28, MK-3475, and a combination of the two. (10D) Bar graph showing the survival and proliferation of cancer cell SCC-25 cultured alone, together with IL-6, co-cultured with monocytes, or co-cultured with monocytes and sCD28.

[0124] Fig.11A -B. (11A) Bar graph of IFNγ secretion from isolated CD3 T cells stimulated with anti-CD3 in the presence of constant CD80-Fc levels and titrated soluble CD28. (11B) Isolated PBMCs stimulated with CMV in the presence of constant sCD28 levels and titrated CD80-Fc.

[0125] Fig. 12A -B. (12A-B) Line graphs of tumor volume of H22 cells inoculated in immunocompetent mice treated with anti-PD-1 antibodies without (12A) and with (12B) administration of recombinant mouse CD28.

[0126] Fig.13A-C. (13A) Line graphs showing the binding of antigen to BSA-conjugated CD28 stem region dimer peptide (right) and recombinant human CD28 protein (left) by serial dilution of clone M9. Antigen was immobilized on a maxisorp ELISA plate. A dilution series of clone M9 was performed and bound antibody was detected with donkey anti-mouse IgG (H&L)-HRP and developed with TMB. (13B) Bar graphs of ELISA detection of recombinant human sCD28 (left) and sCD28 shed from human PBMC activated with SEB (right). The ELISA used antibody #3 as a positive control (2 μg / mL, gray bars), an irrelevant antibody M39 as a negative control (10 μg / mL, dark gray bars) and anti-shear antibody M9 (10 μg / mL, black bars). Recombinant CD28 or shed CD28 was detected by using an ELISA kit detection antibody conjugated to HRP (0.5 μg / mL). (13C) Line graph showing the results of ELISA assays of commercially available MAB342, clone 37407, against increasing concentrations of recombinant human CD28-Fc, recombinant human CD28a, recombinant mouse CD28, and dimeric human CD28 stalk region peptide.

[0127] Fig.14A -E. (14A-B) Bar graphs showing sCD28 levels in supernatants from human CD4+ T cells stimulated with PHA (14A) and human PBMCs stimulated with SEB (14B). (14C) Line graphs showing the results of ELISA assays of increasing concentrations of human sCD28 for antibodies #1 and #2 and commercially available CD28.2. (14D) Line graphs showing the results of ELISA assays of increasing concentrations of human sCD28 for each of antibodies #1, #2, and #3. (14E) Line graphs showing the results of ELISA assays of increasing concentrations of mouse sCD28 for each of antibodies #1, #2, and #3.

[0128] Fig.15A-I. (15A-D) FACS histograms showing CD86 binding to cells expressing mCD28 after addition of CD86 alone (red line) or addition of CD86 and (15A) CD28.2, CD86 and (15B) Antibody #1, CD86 and (15C) Antibody #2, CD86 and (15D) Antibody #3, and CD86 and (15E) mIgG control (green line). Only the secondary antibody was added to show unstained cells (black line). (15F-G) Bar graphs show the secretion of interferon gamma (IFNγ) from: (15F) T cells after treatment with 2 μg / mL anti-CD3, and CD28.2 (2.0 μg / mL) or different dilutions of antibody #1-3 (0.1-10 μg / mL, black bars), (15G) PBMCs after SEB stimulation and treatment with CD28.2 (2.0 μg / mL) or different dilutions of antibody #1-3 (0.1-10 μg / mL, black bars), and (15H) T cells treated with CD80-Fc in the presence and absence of varying concentrations of antibody #1-3. (15I) Histogram of antibody #1-3 binding to naive CD3-positive T cells.

[0129] Fig.16A -G. (16A-B) Bar graphs show sCD28 remaining in blood from cancer patients after mixing with beads conjugated to different amounts of (16A) Antibody #1 and (16B) Antibody #2. (16C-G) Bar graphs show sCD28 remaining in blood from (16C) colorectal cancer patients, (16D-E) melanoma patients, (16F) ovarian cancer patients, and (16G) healthy patients after mixing with beads conjugated to Antibody #1, CD80, CD86, and ICOSL. DETAILED DESCRIPTION

[0130] The present invention provides, in some embodiments, agents that bind membrane CD28 (mCD28) and inhibit the protease cleavage of mCD28 and agents that bind soluble CD28 (sCD28) and are neither CD28 agonists nor CD28 antagonists. The present invention further provides agents that degrade sCD28 or cause degradation of sCD28 after binding, or cause it to be cleared from circulation, tissues and / or tumor microenvironments (TME). In some cases, the agent can perform more than one of these tasks. Also provided are methods for treating cancer and improving PD-1 / PD-L1-based immunotherapy, the methods comprising reducing soluble CD28 (sCD28) levels. The agents and methods of the present invention are based on the surprising discovery that a large number of cancer patients have elevated sCD28 levels in their bloodstreams. sCD28 is a known immunomodulator that is sometimes overexpressed in autoimmune diseases. However, to date, highly elevated levels have not been reported in a wide variety of cancers. Furthermore, it was unexpectedly found that sCD28 can inhibit PD-1 / PD-L1 based immunotherapy.Thus, the reduction of sCD28 in the bloodstream of a subject results in a reduction in the deleterious effects of sCD28 on the subject's anti-cancer ability and the effectiveness of immunotherapy.

[0131] The anti-shearing molecules of the present invention have a dual benefit. By blocking the protease cleavage of mCD28, they can maintain a high CD28 content on the cell surface of T cells. This allows rapid and effective T cell activation, which would be impaired if the level of surface CD28 decreases due to shearing. In addition, the reduction in shearing leads to a reduction in sCD28 in the bloodstream of the subject, thereby reducing the deleterious effects of sCD28 on the subject's anti-cancer ability and the effectiveness of immunotherapy.

[0132] Binding to sCD28

[0133] In a first aspect, an agent is provided that binds soluble CD28 (sCD28) and is neither a CD28 agonist nor a CD28 antagonist.

[0134] In some embodiments, CD28 is mammalian CD28. In some embodiments, CD28 is human CD28. In some embodiments, human CD28 comprises or consists of the following amino acid sequence: MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 1). In some embodiments, mature CD28 lacks a signal peptide and comprises the sequence: NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 2).

[0135] In some embodiments, the DNA coding sequence encoding full-length human CD28 comprises the sequence: ATGCTCAGGCTGCTCTTGGCTCTCAACTTATTCCCTTCAATTCAAGTAACAGGAAACAAGATTTTGGTGAAGCAGTCGCCCATGCTTGTAGCGTACGACAATGCGGTCAACCTTAGCTGCAAGTATTCCTACAATCTCTTCTCAAGGGAGTTCCGGGCATCCCTTCACAAAGGACTGGATAGTGCTGTGGAAGTCTGTGTTGTATATGGGAATTACTCCCAGCAGCTTCAGGTTTACTCAAAAACGGGGTTCAACTGTGATGGGAAATTGGGCAATGAATCAGTGACATTCTACCTCCAGAATTTGTATGTTAACCAAACAGATATTTACTTCTGCAAAATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCTGA (SEQ ID NO:3).

[0136] As used herein, sCD28 refers to any CD28 fragment or variant that does not contain a transmembrane domain and therefore cannot be integrated in a membrane. In some embodiments, the CD28 transmembrane domain comprises the amino acid sequence FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 4). In some embodiments, sCD28 is not membrane-bound. In some embodiments, sCD28 is in solution. In some embodiments, sCD28 is CD28 in blood. In some embodiments, sCD28 is CD28 in TME. In some embodiments, sCD28 is CD28 in body fluids. In some embodiments, sCD28 lacks exon 3 of CD28. In some embodiments, sCD28 is a splice variant of an alternative splice derived from splicing exon 3 of CD28. In some embodiments, sCD28 is a splice product from membrane CD28 (mCD28). In some embodiments, sCD28 is a truncated CD28. In some embodiments, sCD28 lacks the cytoplasmic domain of full-length CD28. In some embodiments, sCD28 is a dimeric sCD28. In some embodiments, sCD28 is a monomeric sCD28. In some embodiments, sCD28 is not an alternatively spliced ​​splice variant derived from CD28. In some embodiments, alternative splicing splices out exon 3 of CD28. In some embodiments, sCD28 comprises the amino acid sequence:

[0137] MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGEE (SEQ ID NO: 5). In some embodiments, sCD28 lacks a signal peptide and comprises the sequence:

[0138] NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGEE (SEQ ID NO: 6).

[0139] In some embodiments, the DNA coding sequence encoding human sCD28 comprises the sequence:

[0140] (SEQ ID NO:7).

[0141] In some embodiments, the reagent binds sCD28 and mCD28, and only causes or causes the degradation or removal of sCD28. In some embodiments, the reagent does not bind membrane CD28 (mCD28). As used herein, mCD28 refers to any CD28 that contains a transmembrane domain and can therefore be integrated into the membrane. In some embodiments, the CD28 transmembrane domain comprises the amino acid sequence FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 4). In some embodiments, mCD28 is in the membrane. In some embodiments, mCD28 has passed through the ER and entered the plasma membrane of the cell through the Golgi apparatus. In some embodiments, mCD28 is in the plasma membrane of immune cells. In some embodiments, mCD28 is in the plasma membrane of T cells.

[0142] In some embodiments, the agent is not a CD28 agonist. In some embodiments, the agent is not a CD28 antagonist. In some embodiments, the agent is neither a CD28 agonist nor a CD28 antagonist. In some embodiments, the sCD28 binding agent is also an mCD28 agonist.

[0143] The term "agonist" generally refers to a molecule, compound or reagent that binds to a receptor and fully or partially activates a receptor. In some embodiments, an agonist binds at the same site as a natural ligand. In some embodiments, an agonist binds at an allosteric site that is different from the binding site of a natural ligand. The term "antagonist" generally refers to a molecule, compound or reagent that binds to a receptor at the same site or another site as an agonist, does not activate a receptor and performs one or more of the following: interferes with or blocks the activation of a natural ligand to a receptor, and interferes with or blocks the activation of a receptor agonist to a receptor. In some embodiments, the antibodies of the present invention bind to mCD28, but do not activate or block the activation of a receptor. In some embodiments, they do not block CD86 activation. In some embodiments, the antibodies of the present invention do not bind to mCD28.

[0144] As used herein, "direct agonist / antagonist" refers to a molecule that binds to a receptor (mCD28) and increases / reduces the signaling of the molecule by binding. In the case of mCD28, the agonist will bind to mCD28 and increase mCD28 signaling in the cell by 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 proinflammatory cytokines. Proinflammatory cytokines are well known in the art and are known to be secreted by activated T cells. Examples of proinflammatory cytokines include, but are not limited to, TNFα, IFNγ, IL-1B, and IL-6. In some embodiments, the proinflammatory cytokine is IFNγ. In the case of mCD28, the antagonist will bind to mCD28 and reduce mCD28 signaling in the cell by binding. In some embodiments, the antagonist reduces T cell activation, reduces T cell proliferation, and / or reduces proinflammatory cytokine secretion. Such molecules are not considered direct agonists / antagonists: molecules that affect the signaling of a receptor by contacting its ligand, contacting an inhibitor, contacting a co-receptor, or contacting any other molecule other than the receptor in question to modify receptor signaling. In some embodiments, the agents of the invention contact sCD28 in serum, thereby allowing increased signaling through mCD28 on cells. Although the result is increased mCD28 signaling, the antibody is not an mCD28 agonist or direct agonist because its binding to mCD28 does not increase receptor signaling.

[0145] In some embodiments, the agent does not bind to the ligand binding domain of mCD28. In some embodiments, the agent does not obscure or prevent access to the ligand binding domain. In some embodiments, the agent does not bind, obstruct or prevent access to the IgV domain of sCD28. In some embodiments, the IgV domain is a ligand binding domain. In some embodiments, the ligand binding domain comprises amino acids 28-137 of SEQ ID NO: 1. In some embodiments, the ligand binding domain comprises or consists of the following amino acid sequence: MLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFN CDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKG (SEQ ID NO: 8). In some embodiments, the agent does not inhibit the binding of sCD28 to the ligand. In some embodiments, the CD28 ligand is selected from: CD80, CD86 and ICOSL. In some embodiments, the CD28 ligand is CD86. In some embodiments, the CD28 ligand is CD80. In some embodiments, the CD28 ligand is ICOSL. In some embodiments, CD86 is CD86-Fc. In some embodiments, CD80 is CD80-Fc.

[0146] In some embodiments, the agent binds to the stem region of CD28. In some embodiments, the agent binds to the membrane proximal region of mCD28. In some embodiments, the stem region comprises the sequence GKHLCPSPLFPGPSKP (SEQ ID NO: 9). In some embodiments, the stem region comprises the sequence KGKHLCPSPLFPGPS (SEQ ID NO: 36). In some embodiments, the stem region comprises or consists of the following sequence: HVKGKHLCPSPLFPGPSKP (SEQ ID NO: 10). In some embodiments, the agent binds to monomeric sCD28. In some embodiments, the agent binds to dimeric sCD28. In some embodiments, the agent binds to monomeric sCD28, dimeric sCD28, or both. In some embodiments, the agent binds to monomeric CD28 but does not bind to dimeric CD28. Since functional mCD28 is dimeric, it can be used to bind only CD28 monomers to ensure that the agent does not bind to mCD28.

[0147] Examples of agents include, but are not limited to, antibodies, antigen-binding fragments of antibodies, nanobodies, single-chain antibodies, single-domain antibodies, small molecules, peptides, and DARPins. In some embodiments, agents are selected from antibodies, antigen-binding fragments of antibodies, Fab fragments, nanobodies, single-chain antibodies, single-domain antibodies, small molecules, peptides, and DARPins. In some embodiments, agents are selected from antibodies, antigen-binding fragments of antibodies, Fab fragments, single-chain antibodies, single-domain antibodies, small molecules, and peptides that specifically bind to CD28. In some embodiments, the agent is a single-domain antibody. In some embodiments, the agent is a nanobody. In some embodiments, the agent is a VHH antibody. As used herein, the terms "single-domain antibody," "nanobody," and "VHH antibody" are synonymous and can be used interchangeably. In some embodiments, the peptide specifically binds to CD28. In some embodiments, the agent is a peptide that has specific binding to CD28. In some embodiments, the peptide is selected from antibodies, antigen-binding fragments of antibodies, Fab fragments, single-chain antibodies, single-domain antibodies, nanobodies, VHH antibodies, and antibody mimetics. As used herein, the term "antibody mimics" refers to organic compounds that can specifically bind to a target antigen. In some embodiments, antibody mimics are structurally unrelated to antibodies. Examples of antibody mimics include, but are not limited to, affilins, affimers, affitins, alpha antibodies, anticalins, avimers, DARPins, fynomers, Kunitz domain peptides, monoclonal antibodies, and nano-CLAMPS. In some embodiments, the antibody mimic is a DARPin. All of these agents are well known in the art and are known to be useful for blocking interactions between receptors and their ligands. Small molecules and proteins that can bind to mCD28 can block cleavage sites or can impair the entry of proteases. In some embodiments, the protein is an antibody mimic. As used herein, the term "DARPin" refers to a designed ankyrin repeat protein. DARPin is a genetically engineered antibody mimic protein that is typically highly specific to its protein target. Therefore, DARPin for CD28 can be an example of an agent.

[0148] In some embodiments, the reagent that binds sCD28 is an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody against sCD28 is a single domain antibody. In some embodiments, the antibody against sCD28 lacks an Fc domain. In some embodiments, the reagent that binds sCD28 is an antigen-binding domain lacking an Fc domain. In some embodiments, the reagent that binds sCD28 is a single domain antibody. In some embodiments, the reagent that binds sCD28 is a camelid, a shark, or a nanobody. In some embodiments, the antibody or fragment is fused to another protein or a fragment of the protein. In some embodiments, the second protein or fragment increases the half-life, particularly the half-life in serum. In some embodiments, the protein that extends the half-life is human serum albumin. In some embodiments, the reagent is modified by a modified chemical that produces an increased half-life. In some embodiments, the modification is PEGylation, and the chemical is polyethylene glycol. The technician will understand that any protein or chemical reagent that extends the half-life or a modification known in the art can be used.

[0149] As used herein, the term "antibody" refers to a polypeptide or polypeptide group comprising at least one binding domain, which is formed by folding a polypeptide chain with a three-dimensional binding space, the internal surface shape and charge distribution of which are complementary to the characteristics of the antigenic determinant of the antigen. Antibodies usually have a tetrameric form, including two pairs of identical polypeptide chains, each pair having a "light" chain and a "heavy" chain. The variable region of each light / heavy chain pair forms an antibody binding site. Antibodies can be oligoclonal antibodies, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, camelid antibodies, CDR-grafted antibodies, multispecific antibodies, bispecific antibodies, catalytic antibodies, humanized antibodies, fully human antibodies, anti-idiotypic antibodies, and antibodies and fragments that can be labeled in soluble form or in a combined form, including epitope binding fragments, variants or derivatives thereof, alone or in combination with other amino acid sequences. Antibodies can be from any species. The term antibody also includes binding fragments, including but not limited to Fv, Fab, Fab', F(ab')2 single-chain antibodies (svFC), dimer variable regions (diabodies) and disulfide-linked variable regions (dsFv). Specifically, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing antigen binding sites. Antibody fragments may or may not be fused to another immunoglobulin domain, including but not limited to the Fc region or a fragment thereof. The skilled person will further appreciate that other fusion products may be produced, including but not limited to scFv-Fc fusions, variable regions (e.g., VL and VH) to Fc fusions, and scFv-scFv-Fc fusions.

[0150] The immunoglobulin molecule can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). In some embodiments, the antibody comprises IgG2 or IgG4. In some embodiments, the antibody comprises IgG2. In some embodiments, the antibody comprises IgG4.

[0151] The basic unit of naturally occurring antibody structure is a heterotetrameric glycoprotein complex of about 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains, which are linked together by non-covalent association and by disulfide bonds. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. There are five human antibody classes (IgG, IgA, IgM, IgD and IgE), and within these classes, various subclasses are recognized based on structural differences (such as the number of immunoglobulin units in a single antibody molecule, the disulfide bond structure of the independent units, and the differences in chain length and sequence). The class and subclass of an antibody are its isotype.

[0152] The amino terminal regions of heavy and light chains are more diverse in sequence than the carboxyl terminal regions, and are therefore referred to as variable domains. This part of the antibody structure confers the antigen binding specificity of the antibody. The heavy chain variable (VH) domain and the light chain variable (VL) domain together form a single antigen binding site, and therefore, the basic immunoglobulin unit has two antigen binding sites. It is believed that specific amino acid residues form an interface between the light chain and heavy chain variable domains (Chothia et al., J. Mol. Biol. 186, 651-63 (1985); Novotny and Haber, (1985) Proc. Natl. Acad. Sci. USA 824592-4596).

[0153] The carboxyl-terminal portions of the heavy and light chains form the constant domains, CH1, CH2, CH3, CL. Although these domains are much less diverse, they vary from one animal species to another, and within the same individual, there are several different antibody isotypes, each with different functions.

[0154] 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 in the hypervariable region defined herein. As used herein, the term "hypervariable region" refers to the amino acid residues in the variable domain of an antibody that are responsible for antigen binding. The hypervariable region contains amino acid residues from the "complementarity determining region" or "CDR". The CDR is primarily responsible for binding to the epitope of the antigen. The scope of FR and CDR has been precisely defined (see Kabat et al.).

[0155] Immunoglobulin variable domains can also be identified using the IMGT information system (www: / / imgt.cines.fr / ) ( / V-Quest) to identify variable region segments, including CDRs. See, e.g., Brochet, X. et al., Nucl. Acids Res. J6: W503-508 (2008).

[0156] Chothia et al. also defined a numbering system applicable to the variable domain sequences of any antibody. One of ordinary skill in the art can clearly assign (assign) any variable domain sequence using the "Chothia numbering" system without any experimental data other than the sequence itself. As used herein, "Chothia numbering" refers to the numbering system proposed by Chothia et al., Journal of Molecular Biology, "Canonical Structures for the Hypervariableregions of immunoglobulins" (1987) and Chothia et al., Nature, "Conformations ofImmunoglobulin Hypervariable Regions" (1989).

[0157] As used herein, the term "humanized antibody" refers to an antibody from a non-human species whose protein sequence has been modified to improve similarity to a human antibody. Humanized antibodies can be produced by recombinant DNA that produces CDRs of non-human antibodies that are surrounded by sequences similar to human antibodies. In some embodiments, humanized antibodies are chimeric antibodies. In some embodiments, humanization includes inserting CDRs of the present invention into a human antibody support or skeleton. Humanized antibodies are well known in the art, and any method for producing these antibodies that retains CDRs of the present invention can be used.

[0158] As used herein, the term "monoclonal antibody" or "mAb" refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for possible variants that may occur during the production of the monoclonal antibody, which variants are usually present in small amounts, the individual antibodies constituting the population are all identical and / or bind the same epitope. Unlike polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates that the characteristic of the antibody is obtained from a substantially homogeneous antibody population, and should not be interpreted as being produced by any particular preparation method. The monoclonal antibodies used according to 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). "Monoclonal antibodies" can also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0159] mAh of the present invention can be any immunoglobulin class including IgG, IgM, IgD, IgE or IgA. The hybridoma producing mAh can be cultured in vitro or in vivo. High titer mAb can be obtained in vivo production, and cells from a separate hybridoma are injected intraperitoneally into pristine-primed Balb / c mice in vivo production to produce ascites containing high concentrations of required mAb. Column chromatography well known to those skilled in the art can be used to purify mAb of isotype IgM or IgG from such ascites or from culture supernatant.

[0160] "Antibody fragments" include a portion of an intact antibody, preferably including its antigen binding region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; tandem diabodies (taDb), linear antibodies (e.g., U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); and one-armed antibodies, single variable domain antibodies, minibodies, single-chain antibody molecules; multispecific antibodies formed by antibody fragments (e.g., including but not limited to Db-Fc, taDb-Fc, taDb-CH3, (scFV)4-Fc, di-scFv, bi-scFv, or tandem (bi, tri)-scFv); and bispecific T cell engagers (BiTEs).

[0161] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields the F(ab')2 fragment, which has two antigen-binding sites and is still capable of cross-linking antigen.

[0162] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and antigen binding site. This region is composed of a dimer (non-covalent association) of a heavy chain variable domain and a light chain variable domain in a tight manner. It is in this configuration that the three surfaces of the VH-VL dimer. The six hypervariable regions together give the antibody antigen binding specificity. However, even if a single variable domain (or only half of an Fv containing three hypervariable regions specific for an antigen) has the ability to recognize and bind to an antigen, its affinity is lower than that of the entire binding site.

[0163] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that some residues are added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation for Fab' herein, wherein the cysteine ​​residue(s) of the constant domains carry at least one free thiol group. F(ab')2 antibody fragments were originally produced as paired Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0164] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.

[0165] According to the amino acid sequence of the constant domain of the heavy chain of the antibody, the antibody can be designated as different classes. There are five main classes of complete antibodies: IgA, IgD, IgE, IgG and IgM, and some of them can be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The heavy chain constant domains corresponding to the antibodies of different classes are respectively referred to as α, δ, e, γ and μ. The subunit structure and three-dimensional configuration of the immunoglobulins of different classes are well known.

[0166] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form a desired antigen-binding structure. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0167] The term "diabody" refers to a mini antibody fragment with two antigen binding sites, which comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, these domains are forced to pair with the complementary domains of another chain and generate two antigen binding sites. Diabody production is known in the art and is described in Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

[0168] The term "multispecific antibody" is used in the broadest sense and particularly encompasses antibodies with multi-epitope specificity. Such multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VHVL unit has multi-epitope specificity; antibodies having two or more VL and VH domains, each of which VHVL units binds to a different epitope; antibodies having two or more single variable domains, each of which binds to a different epitope; full-length antibodies; antibody fragments, such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies, triantibodies, trifunctional antibodies, antibody fragments that have been covalently or non-covalently linked. "Multi-epitope specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different targets.

[0169] The monoclonal antibodies of the present invention can be prepared using methods known in the art. Examples include various techniques such as those described in Kohler, G. and Milstein, C, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); Cole et al., pg.77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

[0170] In addition to the conventional methods of producing antibodies in vivo, phage display technology can also be used to produce antibodies in vitro. Compared with conventional antibody production, the production of this recombinant antibody is much faster, and this recombinant antibody can be produced for a large number of antigens. In addition, when using conventional methods, many antigens are shown to be non-immunogenic or highly toxic, and therefore cannot be used to produce antibodies in animals. In addition, the affinity maturation (i.e., improving affinity and specificity) of recombinant antibodies is very simple and relatively fast. Finally, a large number of different antibodies for specific antigens can be produced in a selection procedure. In order to produce recombinant monoclonal antibodies, people can use various methods based on display libraries to produce a large number of antibodies with different antigen recognition sites. Such libraries can be prepared in several ways: people can produce synthetic libraries by cloning synthetic CDR3 regions in the heavy chain germline gene library, thereby producing a large number of antibody libraries, from which recombinant antibody fragments with various specificities can be selected. People can use human lymphocyte libraries as the starting material for constructing antibody libraries. Naive repertoires of human IgM antibodies can be constructed to create a human library with great diversity. This method has been widely and successfully used to select large numbers 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.

[0171] Non-human antibodies can be humanized by any method known in the art. In one method, non-human complementary determining regions (CDRs) are inserted into human antibodies or consensus antibody framework sequences. Further changes can then be introduced into the antibody framework to adjust affinity or immunogenicity.

[0172] 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.

[0173] In some embodiments, the invention provides nucleic acid sequences encoding the antibodies or antigen-binding portions of the invention.

[0174] 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 will generally include three constant domains: CH1, CH2, and CH3; and a "hinge" region. In some cases, it is desirable to have a constant region.

[0175] Other polypeptides that can be encoded by polynucleotides include antigen-binding antibody fragments, such as single domain antibodies ("dAb"), Fv, scFv, Fab' and CHI and CK or CL domains have been removed. Since mini antibodies are smaller than conventional antibodies, they should achieve better tissue permeability in clinical / diagnostic applications, but since they are bivalent, they should retain higher binding affinity compared to monovalent antibody fragments (such as dAb). Therefore, unless the context indicates otherwise, the term "antibody" used herein not only covers complete antibody molecules, but also covers antigen-binding antibody fragments of the types discussed above. Relative to the corresponding human receptor framework, each framework region present in the encoded polypeptide may include at least one amino acid substitution. Thus, for example, relative to the receptor framework region, 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. Taking into account the characteristics of the individual amino acids constituting the disclosed protein product, those skilled in the art will recognize some reasonable substitutions. Amino acid 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, ie, "conservative substitutions."

[0176] Suitably, the polynucleotides described herein may be isolated and / or purified.In some embodiments, the polynucleotide is an isolated polynucleotide.

[0177] As used herein, the term "non-naturally occurring" substances, ingredients, entities and / or any combination of substances, ingredients, or entities, or any grammatical variations thereof, is a conditional term that expressly excludes, but only excludes, those substances, ingredients, entities and / or any combination of substances, ingredients or entities that are known to one of ordinary skill in the art as "naturally occurring" or that are determined or interpreted by a judge or administrative or judicial body as possibly "occurring in nature" at any time.

[0178] In some embodiments, the agent is an antibody or an antigen-binding portion thereof, comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein: CDR-H1 comprises the amino acid sequence shown in SEQ ID NO:12 (GYTLTNY), CDR-H2 comprises the amino acid sequence shown in SEQ ID NO:13 (NTYTGK), CDR-H3 comprises the amino acid sequence shown in SEQ ID NO:14 (GDANQQFAY), CDR-L1 comprises the amino acid sequence shown in SEQ ID NO:15 (KASQDINSYLS), CDR-L2 comprises the amino acid sequence shown in SEQ ID NO:16 (RANRLVD), and CDR-L3 comprises the amino acid sequence shown in SEQ ID NO:17 (LQYDEFPPT).

[0179] In some embodiments, the agent is an antibody or an antigen-binding portion thereof, comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein: CDR-H1 comprises the amino acid sequence shown in SEQ ID NO:18 (GYTFTSY), CDR-H2 comprises the amino acid sequence shown in SEQ ID NO:19 (YPGDGD), CDR-H3 comprises the amino acid sequence shown in SEQ ID NO:20 (NYRYSSFGY), CDR-L1 comprises the amino acid sequence shown in SEQ ID NO:21 (KSSQSLLNSGNQKNYLT), CDR-L2 comprises the amino acid sequence shown in SEQ ID NO:22 (WASTRES), and CDR-L3 comprises the amino acid sequence shown in SEQ ID NO:23 (QSDYSYPLT).

[0180] In some embodiments, the agent is an antibody or an antigen-binding portion thereof, comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein: CDR-H1 comprises the amino acid sequence shown in SEQ ID NO:24 (GYTFTDY), CDR-H2 comprises the amino acid sequence shown in SEQ ID NO:25 (NPNYDS), CDR-H3 comprises the amino acid sequence shown in SEQ ID NO:26 (SSPYYDSNHFDY), CDR-L1 comprises the amino acid sequence shown in SEQ ID NO:27 (SARSSINYMH), CDR-L2 comprises the amino acid sequence shown in SEQ ID NO:28 (DTSKLAS), and CDR-L3 comprises the amino acid sequence shown in SEQ ID NO:29 (HQRNSYPFT).

[0181] On the other hand, an antibody or an antigen-binding portion thereof is provided, comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein: CDR-H1 comprises the amino acid sequence shown in SEQ ID NO:12 (GYTLTNY), CDR-H2 comprises the amino acid sequence shown in SEQ ID NO:13 (NTYTGK), CDR-H3 comprises the amino acid sequence shown in SEQ ID NO:14 (GDANQQFAY), CDR-L1 comprises the amino acid sequence shown in SEQ ID NO:15 (KASQDINSYLS), CDR-L2 comprises the amino acid sequence shown in SEQ ID NO:16 (RANRLVD), and CDR-L3 comprises the amino acid sequence shown in SEQ ID NO:17 (LQYDEFPPT).

[0182] On the other hand, an antibody or an antigen-binding portion thereof is provided, comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein: CDR-H1 comprises the amino acid sequence shown in SEQ ID NO:18 (GYTFTSY), CDR-H2 comprises the amino acid sequence shown in SEQ ID NO:19 (YPGDGD), CDR-H3 comprises the amino acid sequence shown in SEQ ID NO:20 (NYRYSSFGY), CDR-L1 comprises the amino acid sequence shown in SEQ ID NO:21 (KSSQSLLNSGNQKNYLT), CDR-L2 comprises the amino acid sequence shown in SEQ ID NO:22 (WASTRES), and CDR-L3 comprises the amino acid sequence shown in SEQ ID NO:23 (QSDYSYPLT).

[0183] On the other hand, an antibody or an antigen-binding portion thereof is provided, comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein: CDR-H1 comprises the amino acid sequence shown in SEQ ID NO:24 (GYTFTDY), CDR-H2 comprises the amino acid sequence shown in SEQ ID NO:25 (NPNYDS), CDR-H3 comprises the amino acid sequence shown in SEQ ID NO:26 (SSPYYDSNHFDY), CDR-L1 comprises the amino acid sequence shown in SEQ ID NO:27 (SARSSINYMH), CDR-L2 comprises the amino acid sequence shown in SEQ ID NO:28 (DTSKLAS), and CDR-L3 comprises the amino acid sequence shown in SEQ ID NO:29 (HQRNSYPFT).

[0184] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the following amino acid sequence: QIQLVQSGPELKKPGETVKISCKASGYTLTNYGMNWVKQAPGKGLKWMGWINTYTGK PTYVDDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARGDANQQFAYWGQGTLVTV S (SEQ ID NO: 41). In some embodiments, the variable region of the heavy chain comprises and / or consists of SEQ ID NO: 41. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the following amino acid sequence: QIQLVQSGPELKKPGETVKISCKASGYTLTNYGMNWVKQAPGKGLKWMGWINTYTGKPTYVDDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARGDANQQFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPA SSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 42). In some embodiments, the heavy chain consists of SEQ ID NO: 42. Antibody #1 as referred to in the present application was sequenced and found to have a heavy chain consisting of SEQ ID NO: 42. The CDRs of this heavy chain determined using the Chothia protocol are SEQ ID NOs: 12-14.

[0185] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the following amino acid sequence: DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYDDMGIYYCLQYDEFPPTFGAGTKLELK (SEQ ID NO: 43). In some embodiments, the variable region of the light chain comprises and / or consists of SEQ ID NO: 43. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the following amino acid sequence: DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYDDMGIYYCLQYDEFPPTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 44). In some embodiments, the light chain consists of SEQ ID NO: 44. Antibody #1 as referred to herein was sequenced and found to have a light chain consisting of SEQ ID NO: 44. The CDRs of this light chain determined using the Chothia protocol are SEQ ID NOs: 15-17.

[0186] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the following amino acid sequence: QVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWIKKRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSTTAYMQLSSLASEDSAVYFCARNYRYSSFGYWGQGTLVTVS A (SEQ ID NO: 45). In some embodiments, the variable region of the heavy chain comprises and / or consists of SEQ ID NO: 45. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the following amino acid sequence: QVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWIKKRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSTTAYMQLSSLASEDSAVYFCARNYRYSSFGYWGQGTLVTVSAAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVD KKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK (SEQ ID NO: 46). In some embodiments, the heavy chain consists of SEQ ID NO: 46. Antibody #2 as referred to in the present application was sequenced and found to have a light chain consisting of SEQ ID NO: 46. The CDRs of this heavy chain determined using the Chothia protocol are SEQ ID NOs: 18-20.

[0187] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the following amino acid sequence: DIVMTQSPSSLTVTAGEKVTLSCKSSQSLLNSGNQKNYLTWYQQKPGQPPQLLIYWASTR ESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQSDYSYPLTFGAGTKLELK (SEQ ID NO: 47). In some embodiments, the variable region of the light chain comprises and / or consists of SEQ ID NO: 47. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the following amino acid sequence: DIVMTQSPSSLTVTAGEKVTLSCKSSQSLLNSGNQKNYLTWYQQKPGQPPQLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQSDYSYPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 48). In some embodiments, the light chain consists of SEQ ID NO: 48. In some embodiments, the light chain consists of SEQ ID NO: 48. Antibody #2 as referred to in the present application was sequenced and found to have a light chain consisting of SEQ ID NO: 48. The CDRs of this light chain determined using the Chothia protocol are SEQ ID NOs: 21-23.

[0188] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence: EVQLQQFGAELVKPGASVKISCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPNYDSTAYNQKFMGKATLTVDKSSNTAYMELRSLTSEDTAVYYCARSSPYYDSNHFDYWGQGTSL TVSS (SEQ ID NO: 49). In some embodiments, the variable region of the heavy chain comprises and / or consists of SEQ ID NO: 49. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the following amino acid sequence: EVQLQQFGAELVKPGASVKISCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPNYDSTAYNQKFMGKATLTVDKSSNTAYMELRSLTSEDTAVYYCARSSPYYDSNHFDYWGQGTSLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAH PASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 50). In some embodiments, the heavy chain consists of SEQ ID NO: 50. Antibody #3 as referred to in the present application was sequenced and found to consist of SEQ ID NO: 50. The CDRs of this heavy chain determined using the Chothia protocol are SEQ ID NOs: 24-26.

[0189] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the following amino acid sequence: QIVLTQSPAIMSASPGEKVTMTCSARSSINYMHWFQQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISNMEAEDAATYYCHQRNSYPFTFGSGTKLEIK (SEQ ID NO: 51). In some embodiments, the variable region of the light chain comprises and / or consists of SEQ ID NO: 51. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the following amino acid sequence: QIVLTQSPAIMSASPGEKVTMTCSARSSINYMHWFQQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISNMEAEDAATYYCHQRNSYPFTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO:52). In some embodiments, the light chain consists of SEQ ID NO:52. Antibody #3 as referred to in the present application was sequenced and found to have a light chain consisting of SEQ ID NO:52. The CDRs of this light chain determined using the Chothia protocol are SEQ ID NOs:27-29.

[0190] In some embodiments, the agent binds as a monomer. In some embodiments, the agent binds as a dimer. In some embodiments, the agent binds as a monomer and / or dimer. In some embodiments, the agent binds as a dimer, but does not bind mCD28. In some embodiments, the agent binds as a dimer, but does not crosslink and / or activate mCD28. In some embodiments, the agent binds as a dimer, but only binds a single molecule of CD28. In some embodiments, the agent binds monomeric CD28. In some embodiments, the agent binds dimeric CD28. In some embodiments, the agent binds monomeric and / or dimeric CD28.

[0191] In some embodiments, the antibody or its antigen-binding fragment is an anti-CD28 antibody. In some embodiments, the target antigen of the antibody is CD28, sCD28, dimeric CD28 and / or dimeric sCD28. In some embodiments, the target antigen of the antibody is CD28, sCD28, monomeric CD28 and / or monomeric sCD28. In some embodiments, the target antigen of the antibody is CD28, sCD28, monomeric CD28, monomeric sCD28, dimeric CD28 and / or dimeric sCD28. In some embodiments, sCD28 or CD28 is monomeric. In some embodiments, sCD28 or CD28 is dimeric. In some embodiments, CD28 or sCD28 is monomeric or dimeric. In some embodiments, the antibody or its antigen-binding fragment is an anti-soluble CD28 (sCD28) antibody. "Anti-CD28 antibody", "antibody that recognizes CD28" or "antibody that resists CD28" is an antibody that binds CD28 with sufficient affinity and specificity. In some embodiments, the antibody has increased binding to CD28 or sCD28. In some embodiments, the antibody has increased binding to sCD28 compared to membrane mCD28. In some embodiments, the antibody has increased binding to sCD28 compared to a commercially available CD28 antibody. In some embodiments, the commercially available CD28 antibody is CD28.2. In some embodiments, the antibody or antigen-binding fragment thereof has specific binding affinity for CD28 or sCD28.

[0192] As used herein, the terms "increased binding affinity" and "greater binding affinity" are interchangeable. In some embodiments, the antibodies or antigen binding portions thereof of the present invention have greater binding affinity for sCD28 than for mCD28. In one embodiment, the greater affinity used herein is 10% greater. In one embodiment, the greater affinity used herein is 30% greater. In one embodiment, the greater affinity used herein is 50% greater. In one embodiment, the greater affinity used herein is 75% greater. In one embodiment, the greater affinity used herein is 100% greater. In one embodiment, the greater affinity used herein is 150% greater. In one embodiment, the greater affinity used herein is 250% greater. In one embodiment, the greater affinity used herein is 500% greater. In one embodiment, the greater affinity used herein is 1,000% greater. In one embodiment, the greater affinity used herein is 1.5 times. In one embodiment, the greater affinity used herein is 2 times. In one embodiment, the greater affinity used herein is 5 times. In one embodiment, the greater affinity used herein is 10 times. In one embodiment, the greater affinity used herein is 50 times. In one embodiment, the greater affinity used herein is 100 times. In one embodiment, the greater affinity used herein is 500 times. In one embodiment, the greater affinity used herein is 1000 times.

[0193] "Antigen" is a molecule or part of a molecule that can elicit the formation of antibodies and be bound by antibodies. An antigen can have one or more than one epitope. The specific reaction mentioned above means that an antigen will react with its corresponding antibody in a highly selective manner, and will not react with the many other antibodies that may be induced by other antigens.

[0194] The term "antigenic determinant" or "epitope" according to the present invention refers to a region of an antigen molecule that specifically reacts with a particular antibody. Peptide sequences derived from epitopes can be used alone or in combination with a carrier moiety to immunize animals and produce additional polyclonal or monoclonal antibodies using methods known in the art.

[0195] In some embodiments, the antibody comprises N-linked glycosylation at N304 of the heavy chain.

[0196] In some embodiments, the agent is an antibody or an antigen binding fragment thereof. In some embodiments, the antigen binding fragment is a Fab fragment. In some embodiments, the antibody is a single domain antibody. In some embodiments, the antibody lacks an Fc domain. In some embodiments, the agent is an antigen binding domain lacking an Fc domain. In some embodiments, the agent is a single domain antibody. In some embodiments, the agent is a camelid, a shark, or a 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 half-life, particularly half-life in serum. In some embodiments, the protein that extends half-life is human serum albumin. In some embodiments, the agent is modified by a modified chemical that produces an increased half-life. In some embodiments, the modification is PEGylation, and the chemical is polyethylene glycol. The skilled person will appreciate that any protein or chemical agent that extends half-life or modifications known in the art may be used.

[0197] In some embodiments, the combination of an agent and sCD28 degrades sCD28. In some embodiments, the combination of an agent and sCD28 causes or causes sCD28 degradation. In some embodiments, when the combination is in an organism, degradation occurs. In some embodiments, when the combination is in the bloodstream of an organism, degradation occurs. In some embodiments, when the combination is in the TME of an organism, degradation occurs. In some embodiments, degradation includes removing sCD28 from the blood. In some embodiments, degradation includes transporting the bound sCD28 to a lysosome, endosome, proteasome, or a combination thereof. Each possibility represents a separate embodiment of the present invention.

[0198] In some embodiments, the combination of an agent with sCD28 in an organism results in the removal of sCD28 from the blood. In some embodiments, the combination of an agent with sCD28 in an organism results in the removal of sCD28 from the blood (sweeping). In some embodiments, the combination of an agent with sCD28 in an organism results in the removal of sCD28 from the TME. In some embodiments, the combination of an agent with sCD28 in an organism results in the removal of sCD28 from the TME. In some embodiments, the combination of an agent with sCD28 in an organism results in the removal of sCD28 from the blood, TME, or both. In some embodiments, the combination of an agent with sCD28 in an organism results in the removal of sCD28 from the blood, TME, or both. In some embodiments, sCD28 is not degraded, but is removed from the blood, TME, or both. In some embodiments, the combined sCD28 is an immune complex.

[0199] There are a variety of known mechanisms for removing bound antigens from the bloodstream. These mechanisms include, but are not limited to, complement-mediated removal, phagocytosis of monocytes and macrophages, opsonization, proteolysis, passive diffusion, and active transport. In some embodiments, active transport is by red blood cells. In some embodiments, bound antigens are transported to phagocytic cells. In some embodiments, bound antigens are transported to lysosomes. In some embodiments, bound antigens are transported to lysosomes, endosomes, proteasomes, or combinations thereof. Any reagent that can induce removal of bound sCD28 complexes can be used in the present invention.

[0200] In some embodiments, the agent reduces sCD28 levels by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97 or 99%. Each possibility represents a separate embodiment of the present invention. In some embodiments, the agent reduces sCD28 levels to the level of a healthy individual. In some embodiments, the agent reduces sCD28 levels to at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 ng / ml. Each possibility represents a separate embodiment of the present invention. In some embodiments, the agent reduces sCD28 blood levels to at most 5 ng / ml. In some embodiments, the agent reduces sCD28 blood levels to at most 10 ng / ml. In some embodiments, the agent reduces sCD28 blood levels to at most 20 ng / ml. In some embodiments, the reagent reduces sCD28 levels to the level of a healthy individual. In some embodiments, the reagent reduces sCD28 levels to less than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 ng / ml. Each possibility represents a separate embodiment of the present invention. In some embodiments, the reagent reduces sCD28 levels to less than 5 ng / ml. In some embodiments, the reagent reduces sCD28 levels to less than 10 ng / ml. In some embodiments, the reagent reduces sCD28 levels to less than 20 ng / ml. In some embodiments, the reduction or reduction occurs in the blood, peripheral blood or TME of the subject. In some embodiments, the reduction or reduction occurs in the blood.

[0201] In some embodiments, sCD28 levels are measured by ELISA. In some embodiments, the ELISA is a sandwich ELISA. In some embodiments, the ELISA is a standardized sandwich ELISA. In some embodiments, the ELISA is a Bender MedSystems ELISA. In some embodiments, the ELISA is a Bender MedSystems ELISA kit BMS290. In some embodiments, the ELISA is performed using the reagents of the present invention.

[0202] In some embodiments, the reagent is a removal antibody. As used herein, the term "removal antibody" refers to any antibody or its antigen-binding fragment that reduces the amount of soluble components from a solution. In some embodiments, the removal antibody does not induce antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the removal antibody does not induce complement-dependent cytotoxicity (CDC). In some embodiments, the removal antibody does not induce ADCC and / or CDC. In some embodiments, the removal antibody comprises an IgG2 or IgG4 domain. In some embodiments, the removal antibody comprises an IgG2 domain. In some embodiments, the removal antibody comprises an IgG4 domain. In some embodiments, the removal antibody comprises an IgG1 or IgG3 that is mutated to reduce cell death mediated by antibody binding. In some embodiments, the mutation mutates the Fc receptor binding domain. In some embodiments, the Fc domain of an antibody is modified or mutated to reduce CDC, ADCC, or both. Fc modification is well known in the art, and any mutation or amino acid change known to reduce antibody-mediated cell killing can be used.

[0203] In some embodiments, the antibody does not include IgG1 and / or IgG3. In some embodiments, the antibody does not induce antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the antibody does not induce complement-dependent cytotoxicity (CDC). In some embodiments, the antibody includes an IgG1 or IgG3 containing a mutation that reduces ADCC, CDC or both induced by the combination of the antibody. In some embodiments, the mutation reduces ADCC, CDC or both to nothing. ADCC and CDC have been well characterized, and it is known that the antibody sequences that allow induction of these cytotoxic pathways. Mutation, such as, as a non-limiting example, it is known that IgG1 or IgG3 mutates into IgG2 or IgG4. Any such mutation can be used for the skeleton of the antibody of the present invention.

[0204] In some embodiments, the Fc domain of the antibody is a human Fc domain. In some embodiments, the Fc domain comprises a mutation that reduces ADCC, CDC or both. In some embodiments, the antibody comprises a mutation that increases the dissociation of the antibody or its antigen-binding portion from sCD28 at low pH. In some embodiments, low pH is equal to or lower than 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5, 4.5, 4, 3.5 or 3 pH. Each possibility represents a separate embodiment of the present invention. In some embodiments, low pH is 6 or a pH lower than 6. In some embodiments, low pH is a pH found in human endosomes. In some embodiments, low pH is a pH found in human lysosomes. In some embodiments, the antibody comprises a mutation that increases the dissociation of the antibody or its antigen-binding portion from sCD28 at low calcium concentrations. In some embodiments, the low calcium concentration is a calcium concentration equal to or less than 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.07, 0.05, 0.03 or 0.01 mM. Each possibility represents a separate embodiment of the present invention. In some embodiments, the low calcium concentration is the calcium concentration found in human endosomes. In some embodiments, the mutation that increases dissociation is a mutation in a CDR. In some embodiments, the antibody comprises a mutation that increases the dissociation of the antibody from sCD28 in endosomes and / or lysosomes.

[0205] In some embodiments, the mutation is in the FcRn binding region. In some embodiments, the mutation is in the FcγRIIb binding region. In some embodiments, the mutation increases binding to an Fc receptor. In some embodiments, the Fc receptor is FcRn. In some embodiments, the Fc receptor is FcγRIIb. In some embodiments, the mutation increases Fc receptor binding at neutral pH and / or in serum. In some embodiments, the mutation increases Fc receptor binding at low pH. In some embodiments, the mutation is selected from amino acid 27 of the heavy chain, amino acid 31 of the heavy chain, amino acid 32 of the light chain, and amino acid 53 of the light chain to histidine. In some embodiments, the mutation increases the uptake of the antibody and its bound antigen into cells.

[0206] Examples of mutations that increase Fc receptor binding at low pH, increase uptake into cells, increase dissociation, and / or are used to generate clearance antibodies are well known in the art. Such examples can be found in, for example, Schroter et al., A generic approach to engineer antibody pH-switches using combinatorial histidinescanning libraries and yeast display, mAbs, 2015; Yang et al., Maximizing in vivo target clearance by design of pH-dependent target binding antibodies with altered affinity to FcRn, mAbs, 2017; and Igawa et al., Sweeping antibody as a novel therapeutic antibody modality capable of eliminating soluble antigens from circulation, Immunological Reviews, 2016 (which are all incorporated herein by reference in their entirety). Methods for generating such antibodies and testing their efficacy are also provided. In some embodiments, the mutation is a mutation of a human Fc domain and is selected from mutations of histidine 268 to glutamine, valine 309 to leucine, alanine 330 to serine, and proline 331 to serine. The numbers provided are for IgG1, but comparable mutations such as can be determined by the skilled artisan can be made in other IgGs. In addition, the efficacy of such antibodies can be checked by binding / dissociation assays in culture media at various pHs. Testing can also be performed in model organisms such as mice and monkeys, where serum levels of sCD28 are measured before and after addition of the antibody. Optionally, tumor xenografts expressing human sCD28 can be transplanted into animals to ensure serum sCD28.

[0207] In some embodiments, the antibody comprises a mutation in a CDR that reduces binding to an antigen at low pH. In some embodiments, the antigen is sCD28. In some embodiments, the mutation is a mutation to histidine. In some embodiments, at least 1, 2, 3, 4, or 5 amino acids are mutated to histidine. Each possibility represents a separate embodiment of the present invention. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids are mutated to histidine. Each possibility represents a separate embodiment of the present invention. In some embodiments, an amino acid outside a CDR is mutated to histidine.

[0208] It has been shown that incorporation of histidine residues into the binding interface of antibodies can be used to engineer pH-dependent binding. The basis for pH-sensitive binding comes from the sensitivity of histidine to protonation, which is a result of reduced pH in various microenvironments (including intracellular microenvironments, more specifically endosomal vesicles, more preferably early endosomes). Protonation of histidine side chains in the binding interface can alter electrostatic interactions or induce conformational changes, leading to pH-dependent differences in binding affinity.

[0209] Incorporating pH sensitivity into the antigen binding site can increase the number of antigen binding cycles. pH-dependent antibodies bind with similar high affinity or reduced sufficient affinity to their antigen at plasma pH (pH 7.4) and show reduced binding at low pH, resulting in faster and increased dissociation of the antibody from its antigen binding site in acidic endosomes, thereby enabling recycling back to the plasma and increasing the clearance of the antigen from the plasma.

[0210] His scanning is a mature technique known in the art, by which each amino acid in a CDR is replaced by histidine and pH-dependent binding is assessed. More than one histidine mutation can be combined to synergistically or linearly increase the effect of the mutation. Combinatorial his scanning library methods using phage or yeast display are also well known in the art.

[0211] In some embodiments, the antibody is a clearance antibody. In some embodiments, the antibody is a diagnostic antibody. In some embodiments, the antibody is a therapeutic antibody. In some embodiments, the antibody is an anti-cancer antibody.

[0212] In some embodiments, the agent is a non-antibody protein. In some embodiments, the peptide is a non-antibody peptide. In some embodiments, the non-antibody protein is an antibody mimetic. In some embodiments, the peptide is a polypeptide. In some embodiments, the agent is a small molecule. In some embodiments, the agent is a nucleic acid molecule. In some embodiments, the agent is a synthetic peptide. In some embodiments, the agent is a synthetic binding protein. In some embodiments, the synthetic peptide is based on a non-antibody scaffold. In some embodiments, the agent is an antibody mimetic. In some embodiments, the molar mass of the antibody mimetic is less than 100, 90, 80, 70, 60, 50, 40, 30 or 20 kDa. Each possibility represents a separate embodiment of the present invention. In some embodiments, the agent is a nucleic acid aptamer. In some embodiments, the aptamer is DNA. In some embodiments, the aptamer is RNA. In some embodiments, the aptamer is DNA or RNA.

[0213] In some embodiments, the antibody is used to treat and / or prevent cancer in a subject in need thereof. In some embodiments, the antibody is used to improve immunotherapy in a subject in need thereof. In some embodiments, immunotherapy is an immunotherapy based on PD1 and / or PD-L1. PD-1 and PD-L1 immunotherapy are well known in the art and include PD-1 and PD-L1 blockade and PD-1 and PD-L1 inhibitors. Some examples include, but are not limited to, pembrolizumab (Keytruda), nivolumab (Opdivo), pidilizumab (pidilizumab), cemiplimab (cemiplimab), atezolizumab (atezolizumab) (Tecentriq), avelumab (avelumab) (Bevancio) and durvalumab (durvalumab) (Imfinzi).

[0214] Blocking the shedding of mCD28

[0215] In another aspect, an agent is provided that binds membrane CD28 (mCD28) and inhibits proteolytic cleavage of mCD28.

[0216] As used herein, inhibition of protease cleavage refers to any reduction of the protease cleavage of mCD28. In some embodiments, inhibition is that cleavage is reduced by 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%. Each possibility represents a separate embodiment of the present invention. In some embodiments, inhibition of protease cleavage maintains the level of mCD28 on immune cells. In some embodiments, inhibition of protease cleavage increases the level of mCD28 on immune cells. In some embodiments, inhibition of protease cleavage maintains the level of mCD28 that is sufficient for immunostimulation.

[0217] In some embodiments, the reduction of protease cleavage is a reduction of the cleavage of at least one protease. In some embodiments, the reduction of protease cleavage is a reduction of the cleavage of at least one metalloprotease. In some embodiments, the metalloprotease is ADAM 10, ADAM 17 or both.

[0218] In some embodiments, the agent is an antibody or antigen binding fragment thereof. In some embodiments, the antibody or fragment is fused to another protein or fragment of a protein. In some embodiments, the second protein or fragment increases half-life, particularly half-life in serum. In some embodiments, the protein that extends half-life is human serum albumin. In some embodiments, the agent is modified by a chemical that produces a modification that increases half-life. In some embodiments, the modification is PEGylation and the chemical is polyethylene glycol. The skilled artisan will appreciate that any protein or chemical agent that extends half-life or modification known in the art may be used.

[0219] In some embodiments, the agent binds as a monomer. In some embodiments, the agent binds as a dimer. In some embodiments, the agent binds as a monomer and / or dimer. In some embodiments, the agent binds as a dimer, but does not crosslink and / or activate mCD28. In some embodiments, the agent binds as a dimer, but only binds a single molecule of CD28. In some embodiments, the agent binds to monomeric CD28. In some embodiments, the agent binds to dimeric CD28. In some embodiments, the agent binds to monomeric and / or dimeric CD28.

[0220] In some embodiments, the agent is not a CD28 agonist. In some embodiments, the agent is not a CD28 antagonist. In some embodiments, the agent is neither a CD28 agonist nor a CD28 antagonist.

[0221] In some embodiments, the agent does not bind to the ligand binding domain of mCD28. In some embodiments, the agent does not hinder or prevent access to the ligand binding domain. In some embodiments, the agent binds to the cleavage site. In some embodiments, the agent hinders or prevents access to the cleavage site. In some embodiments, the agent prevents protease from entering the cleavage site. In some embodiments, the agent binds to the stem region of CD28. In some embodiments, the agent binds to the membrane proximal region of mCD28. In some embodiments, the cleavage site is within the stem region. In some embodiments, the stem region comprises the sequence GKHLCPSPLFPGPSKP (SEQ ID NO: 9). In some embodiments, the stem region comprises the sequence KGKHLCPSPLFPGPS (SEQ ID NO: 36). In some embodiments, the stem region comprises or consists of the following sequence: HVKGKHLCPSPLFPGPSKP (SEQ ID NO: 10).

[0222] 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.

[0223] In some embodiments, the agent does not modulate CD28 function and / or signaling. In some embodiments, the agent does not degrade mCD28. In some embodiments, the agent does not cause or promote mCD28 degradation. In some embodiments, signaling is mCD28-mediated immune cell activation. In some embodiments, the agent does not inhibit immune cell activation. In some embodiments, the agent does not induce CD28 receptor internalization or recycling. Co-stimulation via mCD28 is essential for 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 retained in the membrane. Therefore, the sheared CD28 cannot send signals and cannot promote T cell activation. Therefore, agents that block shearing and are also antagonists do not allow mCD28 activation. Similarly, agents that block shearing but are also agonists may induce abnormal T cell activation and may induce autoimmune responses. In some embodiments, the agent is not the anti-CD28 antibody MAB342. In some embodiments, the agent is not the anti-CD28 antibody clone #37407.

[0224] In some embodiments, the agent does not reduce the surface level of mCD28 on immune cells. In some embodiments, the immune cells are T cells. In some embodiments, the agent reduces the surface level of mCD28 by less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 7%, 5%, 3%, 2% or 1%. Each possibility represents a separate embodiment of the present invention.

[0225] In some embodiments, the combination of the reagent and the cell does not kill the cell. In some embodiments, the combination of the reagent and the cell does not cause 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 an IgG1 or IgG3 that is mutated to reduce cell death mediated by antibody binding. In some embodiments, the mutation mutates the Fc receptor binding domain. In some embodiments, the Fc domain of the antibody is transformed or mutated to reduce CDC, ADCC or both. Fc transformation is well known in the art, and any mutation or amino acid change known to reduce antibody-mediated cell killing can be used.

[0226] In some embodiments, the agent is a non-antibody protein. In some embodiments, the agent is a small molecule. In some embodiments, the agent is a nucleic acid molecule. In some embodiments, the agent is a synthetic peptide. In some embodiments, the agent is a synthetic binding protein. In some embodiments, the synthetic peptide is based on a non-antibody scaffold. In some embodiments, the agent is an antibody mimetic. In some embodiments, the molar mass of the antibody mimetic is less than 100, 90, 80, 70, 60, 50, 40, 30 or 20 kDa. Each possibility represents a separate embodiment of the present invention. In some embodiments, the agent is a nucleic acid aptamer. In some embodiments, the aptamer is DNA. In some embodiments, the aptamer is RNA. In some embodiments, the aptamer is DNA or RNA. Examples of antibody mimics include, but are not limited to, affilins, affimers, affitins, alpha antibodies, anticalins, avimers, DARPins, fynomers, Kunitz domain peptides, monoclonal antibodies, and nano-CLAMPS. In some embodiments, the antibody mimetic is a DARPin.

[0227] In some embodiments, the agent inhibits proteolytic cleavage of 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 ​​protease or a 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 protease, glutamic protease, serine protease, cysteine ​​protease and threonine protease. In some embodiments, the protease is an asparagine peptide cleavage enzyme. In some embodiments, the protease is a shedding enzyme (sheddase). In some embodiments, the metalloprotease is an exopeptidase. In some embodiments, the metalloprotease is an endopeptidase. In some embodiments, the metalloprotease is an exopeptidase or an endopeptidase. In some embodiments, the metalloprotease is zinc catalyzed. In some embodiments, the metalloprotease is cobalt catalyzed. In some embodiments, the metalloprotease is ADAM10. In some embodiments, the metalloprotease is ADAM17. In some embodiments, the metalloprotease is ADAM 10 and / or ADAM 17. In some embodiments, the metalloprotease is ADAM 10, ADAM 17, or both.

[0228] In some embodiments, the antibody or antigen-binding fragment thereof comprises three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein: CDR-H1 comprises the amino acid sequence shown in SEQ ID NO:30 (GFTFSSYYMS), CDR-H2 comprises the amino acid sequence shown in SEQ ID NO:31 (TISDGGDNTYYAGTVTG), CDR-H3 comprises the amino acid sequence shown in SEQ ID NO:32 (IHWPYYFDS), CDR-L1 comprises the amino acid sequence shown in SEQ ID NO:33 (RASSSVSYMN), CDR-L2 comprises the amino acid sequence shown in SEQ ID NO:34 (ATSDLAS), and CDR-L3 comprises the amino acid sequence shown in SEQ ID NO:35 (QQWSSHPPT).

[0229] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the following amino acid sequence: DVKLVESGGGLVKLGGSLKLSCVASGFTFSSYYMSWVRQTPEKRLEWVATISDGGDNTY YAGTVTGRFTISRDFAKNTLYLQMNSLTSEDTAVYYCARIHWPYYFDSWGQGTTLTVSS (SEQ ID NO: 53). In some embodiments, the variable region of the heavy chain comprises and / or consists of SEQ ID NO: 53. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the following amino acid sequence: GACGTGAAGCTCGTGGAGTCTGGGGGAGGCTTAGTGAAGCTTGGAGGGTCCCTGAAACTCTCCTGTGTAGCCTCTGGATTCACTTTCAGTAGCTATTACATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCGACCATAAGTGATGGTGGTGATAACACCTACTACGCAGGCACTGTGACGGGCCGATTCACCATCTCCAGAGACTTTGCCAAGAACACCCTGTACCTGCAAATGAACAGTCTGACCTCTGAGGACACAGCCGTGTATTACTGTGCAAGAATTCATTGGCCTTACTATTTTGACTCCTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO: 54). In some embodiments, the heavy chain consists of SEQ ID NO: 54. The cleavage-resistant antibody of the present application was sequenced and found to have a heavy chain consisting of SEQ ID NO: 54. The CDRs of this heavy chain determined using the Chothia protocol are SEQ ID NOs: 30-32.

[0230] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the following amino acid sequence: QFVLSQSPAILSASPGEMLTMTCRASSSVSYMNWYQQKPGSSPKPWIYATSDLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSHPPTFGGGTKLEIR (SEQ ID NO: 55). In some embodiments, the variable region of the light chain comprises and / or consists of SEQ ID NO: 55. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the following amino acid sequence: CAATTTGTTCTCTCCCAGTCTCCAGCAATCCTGTCTGCATCTCCCGGGGAGATGCTCACAATGACTTGCAGGGCCAGCTCAAGTGTAAGTTATATGAACTGGTATCAGCAGAAGCCAGGATCTTCCCCCAAACCCTGGATTTATGCCACATCCGACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTATTCTCTCACAATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTCACCCACCCACGTTCGGAGGGGGGACCAAGCTGGAAATAAGA (SEQ ID NO: 56). In some embodiments, the light chain consists of SEQ ID NO: 56. The cleavage-resistant antibody as referred to in the present application was sequenced and found to have a light chain consisting of SEQ ID NO: 56. The CDRs of this light chain, determined using the Chothia protocol, are SEQ ID NOs: 33-35.

[0231] Methods of treating / preventing cancer

[0232] In another aspect, provided is a method of treating and / or preventing cancer in a subject in need thereof, the method comprising reducing the level of soluble CD28 (sCD28) in the subject.

[0233] In another aspect, a method of improving immunotherapy in a subject in need thereof is provided, the method comprising reducing sCD28 levels in the subject.

[0234] 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 administration of anti-PD1 or anti-PD-L1 antibodies. In some embodiments, the therapy comprises blockade of the PD-1 checkpoint. In some embodiments, the immunotherapy comprises administration of allogeneic, syngeneic, or autologous immune cells to the subject. In some embodiments, the immune cells are T cells. In some embodiments, the subject in need of immunotherapy has cancer.

[0235] As used herein, the terms "treatment" or "treating" of a disease, disorder, or condition encompass alleviating at least one symptom thereof, reducing its severity, or inhibiting its progression. Treatment does not necessarily mean that the disease, disorder, or condition is completely cured. To be an effective treatment, the compositions useful herein need only reduce the severity of the disease, disorder, or condition, reduce the severity of the symptoms associated therewith, or improve the quality of life of the patient or subject.

[0236] In some embodiments, reducing comprises administering to the subject at least one reagent of the present invention. As used herein, the terms "administering", "administering", etc. refer to any method in reasonable medical practice to deliver a composition comprising an active agent to a subject in a manner that provides a therapeutic effect. One aspect of this theme provides oral administration of a therapeutically effective amount of the reagent of the present invention to a patient in need thereof. Other suitable routes of administration may include parenteral, subcutaneous, intravenous, intramuscular or intraperitoneal.

[0237] In another aspect, a pharmaceutical composition is provided, comprising an agent of the invention and a therapeutically acceptable carrier, adjuvant or excipient. In some embodiments, the administration is administration of a pharmaceutical composition of the invention.

[0238] As used herein, the term "carrier", "excipient" or "adjuvant" refers to any component of a pharmaceutical composition that is not an active agent. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic, inert solid, semi-solid, liquid filler, diluent, encapsulating material, any type of formulation aid, or simply a sterile aqueous medium, such as saline. Some examples of materials that can be used as pharmaceutically acceptable carriers are sugars such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate, powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethanol solutions and phosphate buffered saline solutions, and other nontoxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of materials that can be used as carriers herein include sugars, starches, cellulose and its derivatives, powdered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffered solution, cocoa butter (suppository base), emulsifiers, and other non-toxic pharmaceutically compatible materials used in other pharmaceutical formulations. Wetting agents and lubricants, such as sodium lauryl sulfate, as well as colorants, flavoring agents, excipients, stabilizers, antioxidants, and preservatives may also be present. Any non-toxic, inert, and effective carrier may be used to formulate the compositions contemplated herein. In this regard, suitable pharmaceutically acceptable carriers, excipients and diluents are well known to those skilled in the art, such as those described in The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, NJ (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and the "Inactive Ingredient Guide," US Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management (which are incorporated herein by reference in their entirety).Examples of pharmaceutically acceptable excipients, carriers, and diluents for use in the present compositions include distilled water, saline, Ringer's solution, dextrose solution, Hank's solution, and DMSO. These additional inactive components, 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 edition, Gilman et al. Eds. Pergamon Press (1990); Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21st edition, Lippincott Williams & Wilkins, Philadelphia, Pa., (2005) (each of which is incorporated herein by reference in its entirety). The compositions described herein may also be contained in artificially generated structures such as liposomes, ISCOMS, slow-release particles, and other vehicles that increase the half-life of peptides or polypeptides in serum. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, etc. The liposomes used with the peptides described herein are formed by standard vesicle-forming lipids, which typically include neutral and negatively charged phospholipids and sterols, such as cholesterol. The choice of lipids generally depends on considerations such as liposome size and stability in blood. For example, Coligan, JE et al., Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, review various methods that can be used to prepare liposomes, and also see U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028 and 5,019,369.

[0239] Carriers may comprise in total from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0240] In some embodiments, reduction includes administering an inhibitory nucleic acid molecule that binds to the mRNA encoding sCD28 but not to the mRNA encoding mCD28. A possible source of sCD28 comes from the translation of CD28 transcription variants lacking a transmembrane domain. Such variants can have a unique mRNA sequence that can be targeted to degrade or inhibit mRNA translation. For non-limiting examples, sCD28 is produced by an mRNA splice variant lacking exon 3 (SEQ ID NO:7). In this variant, exon 2 and exon 4 are connected to be present in the variant but lacking in the unique sequence in full-length CD28. In some embodiments, a nucleic acid molecule is bound to at least the connection of exon 2 and exon 4 of SEQ ID NO:7. In some embodiments, the splice connection of exons 2 and 4 includes the sequence AAAGGTGA (SEQ ID NO:11). In some embodiments, the nucleic acid molecule binds to at least 10, 15, 20, 25, 30, 35, 40, 45, or 50 bases of SEQ ID NO: 7, including the splice junction of exons 2 and 4. Each possibility represents a separate embodiment of the invention.

[0241] In some embodiments, the nucleic acid molecule is an siRNA. In some embodiments, the molecule is an shRNA. In some embodiments, the molecule is an siRNA or an shRNA.

[0242] In some embodiments, the reduction includes administering an agent that inhibits a protease that can cleave mCD28. In some embodiments, the agent inhibits ADAM 10, ADAM 17, or both. Protease inhibitors are well known in the art. Examples of protease inhibitors that inhibit ADAM17 and ADAM10 include, but are not limited to, TAPI-1, GM6001, and GI254023X. Other therapeutic protease inhibitors are disclosed in International Patent Application WO2004096139.

[0243] In some embodiments, reducing comprises administering a peptide comprising the stalk region of CD28 or a fragment thereof. In some embodiments, the peptide is monomeric. In some embodiments, the peptide is dimeric. In some embodiments, CD28 is human CD28. In some embodiments, the peptide inhibits protease access to the cleavage site. In some embodiments, the peptide induces the production of autoantibodies that block the cleavage site.

[0244] In some embodiments, the methods of the present invention do not degrade mCD28 or do not cause mCD28 degradation. In some embodiments, the methods of the present invention do not reduce the level of mCD28 on immune cells. In some embodiments, the methods of the present invention do not reduce mCD28-mediated immune cell activation. In some embodiments, the methods of the present invention maintain the level of mCD28 on immune cells in the object. In some embodiments, the methods of the present invention increase the level of mCD28 on immune cells in the object.

[0245] In some embodiments, the reduction is a reduction of sCD28 by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 99%. Each possibility represents a separate embodiment of the present invention. In some embodiments, the reduction is in serum sCD28. In some embodiments, the reduction is in the blood level of sCD28. In some embodiments, the reduction is in the level of sCD28 in the tumor microenvironment (TME).

[0246] In some embodiments, the blood of the subject comprises elevated levels of sCD28. In some embodiments, before the reduction, the blood of the subject comprises elevated levels of sCD28. In some embodiments, the level is elevated to above the level of a healthy subject. In some embodiments, the sCD28 level of the subject is elevated to at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% above the level of a healthy subject. Each possibility represents a separate embodiment of the present invention. In some embodiments, the level is elevated to above 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45 or 50 ng / ml of blood. Each possibility represents a separate embodiment of the present invention. In some embodiments, the level is increased to more than 5 ng / ml. In some embodiments, the level is increased to more than 10 ng / ml. In some embodiments, the level is increased to more than 20 ng / ml. In some embodiments, the blood of the subject comprises at least 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45 or 50 ng sCD28 per milliliter of blood. Each possibility represents a separate embodiment of the present invention. In some embodiments, before the reduction, the blood of the subject comprises at least 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45 or 50 ng sCD28 per milliliter of blood. Each possibility represents a separate embodiment of the present invention. In some embodiments, the blood of the subject comprises at least 5 ng / ml sCD28. In some embodiments, the blood of the subject comprises at least 10 ng / ml sCD28. In some embodiments, the subject's blood comprises at least 20 ng / ml sCD28. In some embodiments, prior to the reduction, the subject's blood comprises at least 5 ng / ml sCD28. In some embodiments, prior to the reduction, the subject's blood comprises at least 10 ng / ml sCD28. In some embodiments, prior to the reduction, the subject's blood comprises at least 20 ng / ml sCD28.

[0247] In some embodiments, the subject has cancer. In some embodiments, the cancer is a cancer that can be treated with PD-1 / PD-L1 therapy. In some embodiments, the subject has undergone PD-1 / PD-L1 therapy. In some embodiments, the subject is unresponsive to PD-1 / PD-L1 therapy. In some embodiments, the subject has not undergone ( In some embodiments, the methods of the present invention are performed together with PD-1 / PD-L1 therapy. In some embodiments, the methods of the present invention are performed before PD-1 / PD-L1 therapy.

[0248] In some embodiments, the method further comprises administering another immunotherapy to the subject. In some embodiments, the method further comprises administering an immunotherapy based on PD-1 and / or PD-L1. In some embodiments, the another 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 another immunotherapy is an immunotherapy based on a chimeric antigen receptor (CAR). In some embodiments, the CAR is a CAR-T. In some embodiments, the CAR is a CAR-NK. In some embodiments, the another immunotherapy is a cancer vaccine.

[0249] As used herein, the terms "CAR-T cells" and "CAR-NK cells" refer to receptors that are specific to at least one target protein (e.g., an immunogenic protein that is expressed after treatment with an epigenetic modifier) ​​and that are transplanted to immune effector cells (T cells or NK cells). In some embodiments, CAR-T cells have the specificity of monoclonal antibodies transplanted to T cells. In some embodiments, CAR-NK cells have the specificity of monoclonal antibodies transplanted to NK cells. In some embodiments, T cells are selected from cytotoxic T lymphocytes and regulatory T cells.

[0250] 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 receptor-bound proteins. In some embodiments, CAR-T or CAR-NK cells target at least one viral protein. In some embodiments, CAR-T or CAR-NK cells target multiple viral proteins. In some embodiments, CAR-T or CAR-NK cells target viral proteins that are enhanced in expression due to contact with epigenetic modifiers.

[0251] The construction of CAR-T cells is well known in the art. In a non-limiting example, monoclonal antibodies against viral proteins can be prepared, and then a vector encoding the antibody will be constructed. The vector will also include a costimulatory signal region. In some embodiments, the costimulatory signal region includes an intracellular domain of a known T cell or NK cell 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, ICQS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand specifically bound to CD83. In some embodiments, the vector also includes a CD3Z signaling domain. This vector is then transfected into T cells, for example, by lentiviral infection.

[0252] In some embodiments, the cancer is a cancer with elevated sCD28 levels. In some embodiments, the cancer comprises high sCD28 levels. In some embodiments, elevated and / or high sCD28 levels are 5, 6, 7, 8, 9, 10, 12, 14, 15, 17, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or 100 ng / ml and / or levels above them. Each may represent a separate embodiment of the present invention. In some embodiments, the cancer comprises high sCD28 levels. In some embodiments, the elevated and / or high sCD28 levels are 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900 or 1000% or more 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, kidney cancer, gastric cancer and colorectal cancer. In some embodiments, the cancer is selected from melanoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, kidney cancer, gastric cancer or colorectal cancer. Each possibility represents a separate embodiment of the present invention.

[0253] In some embodiments, the methods are performed in vivo. In some embodiments, the methods are performed in vitro. In some embodiments, the reduction is performed in vivo. In some embodiments, the reduction is performed in vitro. In some embodiments, the reduction comprises removing blood from the subject, thereby reducing the level of sCD28 in the removed blood and returning the blood to the subject, thereby reducing sCD28 in the subject. Methods of dialysis and blood cleaning are well known. The present invention can be implemented by removing sCD28 in vitro and then returning the blood to the subject.

[0254] How to use

[0255] In another aspect, a method for detecting sCD28 in a sample in vitro is provided, the method comprising:

[0256] a. Providing a sample comprising sCD28;

[0257] b. contacting the sample with a reagent of the present invention; and

[0258] c. detecting a reagent that binds to sCD28;

[0259] Thereby, sCD28 in the sample is detected.

[0260] In some embodiments, the sample is from a subject. In some embodiments, the sample comprises a body fluid. In some embodiments, the sample comprises a tissue. In some embodiments, the sample comprises a cell. In some embodiments, detection is performed by a secondary antibody. In some embodiments, detection is performed by which marker molecule is bound to the reagent. In some embodiments, detection is performed by ELISA. In some embodiments, detection is performed by immunohistochemistry. In some embodiments, detection is performed by immunoblotting. In some embodiments, the reagents of the present invention are used to detect sCD28. In some embodiments, the reagents of the present invention are used to detect sCD28 without detecting mCD28.

[0261] In another aspect, a method of determining the suitability of a subject to be treated by a method of treatment of the invention is provided, the method comprising providing a sample from the subject and determining the level of sCD28 in the sample, wherein an elevated level of sCD28 indicates that the subject is suitable for treatment by a method of treatment of the invention.

[0262] In another aspect, a method for determining the suitability of a subject to be treated by anti-PD-1 and / or PD-L1 immunotherapy and / or by CD80 and / or CD86-based immunotherapy is provided, the method comprising providing a sample from the subject and determining the level of sCD28 in the sample, wherein an sCD28 level above 5 ng / ml indicates that the subject is not suitable for treatment by anti-PD-1 and / or PD-L1 immunotherapy or CD80 and / or CD86-based immunotherapy.

[0263] In another aspect, a method of making an unfit subject fit to receive anti-PD-1 and / or PD-L1 immunotherapy and / or CD80 and / or CD86-based immunotherapy is provided, the method comprising reducing sCD28 levels in the unfit subject, thereby making the subject fit.

[0264] In another aspect, provided is a method of improving CD80 and / or CD86-based immunotherapy in a subject, the method comprising:

[0265] a. measuring the level of sCD28 in a subject; and

[0266] b. increasing the dose of CD80 and / or CD86-based immunotherapy for subjects with sCD28 levels above 5 ng / ml;

[0267] Thereby improving CD80 and / or CD86-based immunotherapy.

[0268] In some embodiments, the method further comprises rendering the unfit subject fit by performing the method of the invention.

[0269] In some embodiments, the elevated level is elevated to levels near healthy subjects. In some embodiments, the elevated level is elevated to levels near a predetermined threshold. In some embodiments, the elevated level is sCD28 levels above 5, 10, 15, 15, 20, 25, 30, 35, 40, 45 or 50 ng / ml. Each possibility represents a separate embodiment of the present invention. In some embodiments, sCD28 levels above 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 ng / ml indicate that the subject is not suitable for treatment. Each possibility represents a separate embodiment of the present invention. In some embodiments, sCD28 levels above 10 indicate that the subject is not suitable for treatment. In some embodiments, sCD28 levels above 20 indicate that the subject is not suitable for treatment.

[0270] CD80 and CD86 immunotherapy is well known in the art and includes administering CD80 / CD86 and or its mimetics, derivatives or mimetics to stimulate an immune response. By way of non-limiting example, CD80-Fc is currently in clinical trials as an anti-cancer immunotherapy.

[0271] In some embodiments, detecting an agent bound to sCD28 comprises determining the amount of bound sCD28. In some embodiments, detecting the amount of bound sCD28 is determining the amount of sCD28 in a body fluid. In some embodiments, the method is used to determine the suitability of an object for treatment with a pharmaceutical composition of the present invention. In some embodiments, sCD28 levels above a predetermined threshold indicate that the object is suitable for treatment with the composition of the present invention. In some embodiments, the method is used to determine the suitability of an object for immunotherapy. In some embodiments, sCD28 levels below a predetermined threshold or the absence of sCD28 indicate that the object is suitable for immunotherapy. In some embodiments, sCD28 levels above a predetermined threshold indicate that the object is suitable for immunotherapy in combination with the composition of the present invention.

[0272] In some embodiments, the subject has cancer. In some embodiments, the subject is at risk of developing cancer.

[0273] In some embodiments, the method further comprises administering to the subject a pharmaceutical composition of the invention. In some embodiments, the method further comprises administering to the subject a pharmaceutical composition of the invention when the detected sCD28 is above a predetermined threshold. In some embodiments, the method further comprises administering to the subject an immunotherapy.

[0274] Reagent test kit

[0275] In another aspect, a kit is provided, comprising at least one agent of the invention or a pharmaceutical composition of the invention.

[0276] 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 stating that the agent of the present invention is used in combination with an immunotherapy based on PD-1 and / or PD-L1. In some embodiments, the kit comprises a label stating that the antibody or pharmaceutical composition of the present invention is used in combination with an immunotherapy based on PD-1 and / or PD-L1.

[0277] In some embodiments, the kit further comprises a detection molecule for detecting the reagent of the present invention. In some embodiments, the detection molecule is a second detection molecule. In some embodiments, the detection molecule is combined with the reagent. Detection molecules are well known in the art and include, but are not limited to, fluorescent moieties and molecules, dyes, and secondary antibodies.

[0278] In another aspect, a kit comprising a PD-1 and / or PD-L1 based immunotherapy is provided, the kit comprising a label stating that the PD-1 and / or PD-L1 based immunotherapy is used in combination with an antibody or pharmaceutical composition of the invention.

[0279] In some embodiments, the kit of the present invention is used to treat 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 suffers from cancer. In some embodiments, the kit of the present invention is used to determine the suitability of a subject to be treated with an agent or pharmaceutical composition of the present invention. In some embodiments, the kit is used to determine the suitability of a subject to be treated with an anti-PD-1 / PD-L1 based immunotherapy.

[0280] Reagent Generation Method

[0281] In another aspect, there is provided a method for producing an agent of the invention, the method comprising:

[0282] a. obtaining a reagent that specifically binds to the extracellular domain of CD28 or a fragment thereof; and

[0283] b. testing the ability of the reagent to block the cleavage of mCD28 by a protease, and selecting at least one reagent that blocks the cleavage of mCD28 by a protease;

[0284] Thereby, the reagent of the present invention is produced.

[0285] In another aspect, a method for producing an agent of the invention is provided, the method comprising:

[0286] Cultivating a host cell containing one or more vectors comprising a nucleic acid sequence encoding an agent, wherein the nucleic acid sequence is a nucleic acid sequence of an agent selected by:

[0287] i. obtaining a reagent that binds to the extracellular domain of CD28 or a fragment thereof;

[0288] ii. testing the ability of the reagent to block cleavage of mCD28 by a protease; and

[0289] iii. selecting at least one reagent that blocks the cleavage of mCD28 by a protease;

[0290] Thereby, the reagent of the present invention is produced.

[0291] In some embodiments, the agent is an anti-shearing agent. In some embodiments, the agent is an anti-shedding agent. In some embodiments, the agent reduces shedding of sCD28 in a subject. In some embodiments, the agent reduces shearing of mCD28. In some embodiments, the agent reduces shearing of mCD28 in a subject.

[0292] In some embodiments, the protease is ADAM 10. In some embodiments, the protease is ADAM 17. In some embodiments, the protease is ADAM 10, ADAM 17, or both.

[0293] As used herein, the term "extracellular domain of CD28" refers to the N-terminal portion of CD28 before the transmembrane domain. In some embodiments, the extracellular domain of CD28 is sCD28. In some embodiments, the extracellular domain of CD28 is CD28a. In some embodiments, the extracellular domain of CD28 is the CD28 stem domain. In some embodiments, the extracellular domain of CD28 comprises the stem domain of CD28. In some embodiments, the extracellular domain of CD28 comprises the sequence NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 37). In some embodiments, the extracellular domain of CD28 or a fragment thereof is a dimer. In some embodiments, the extracellular domain of CD28 or a fragment thereof is monomeric. In some embodiments, the extracellular domain of CD28 or a fragment thereof is dimer or monomeric.

[0294] As used herein, "fragment" refers to a partial polypeptide that constitutes a portion of a larger protein or protein domain. In some embodiments, the fragment comprises at least 10, 20, 30, 40 or 50 amino acids. Each possibility represents a separate embodiment of the present invention. In some embodiments, the fragment comprises at most 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids. Each possibility represents a separate embodiment of the present invention. In some embodiments, the reagent for obtaining a fragment that binds to the extracellular domain of CD28 is a reagent for obtaining a specific binding to the stalk domain of CD28.

[0295] In some embodiments, the method further comprises measuring mCD28 downstream signaling in the presence of the obtained agent, and selecting at least one agent that neither greatly excites nor greatly antagonizes mCD28 signaling. In some embodiments, the selection is to select at least one agent that does not antagonize mCD28 signaling. Those skilled in the art will understand that for cancer treatment, exciting CD28 signaling may not be harmful, but antagonizing signaling will be counterproductive.

[0296] In some embodiments, the ability of the reagent to block shearing is tested by the method described below. In some embodiments, the ability of the test reagent to block shearing includes mixing the reagent, protease and the extracellular domain of CD28 or a fragment thereof containing a shearing site. In some embodiments, the test further includes sequencing the extracellular domain of CD28 or a fragment thereof to check for truncation and / or shearing. In some embodiments, the test further includes running the extracellular domain of CD28 or a fragment thereof on a gel sensitive enough to measure the size changes caused by shearing. In some embodiments, the test further includes measuring the generation of sCD28 from cells expressing mCD28 in the presence of a reagent and a protease.

[0297] In another aspect, a method for producing an agent of the invention is provided, the method comprising:

[0298] a. obtaining an agent that binds to the extracellular domain of CD28 or a fragment thereof; and

[0299] b. measuring mCD28 downstream signaling in the presence of the obtained reagent, and selecting at least one reagent that neither greatly stimulates nor antagonizes mCD28 signaling;

[0300] Thereby, the reagent of the present invention is produced.

[0301] In another aspect, a method for producing an agent of the invention is provided, the method comprising:

[0302] Cultivating a host cell containing one or more vectors comprising a nucleic acid sequence encoding an agent, wherein the nucleic acid sequence is a nucleic acid sequence of an agent selected by:

[0303] i. obtaining a reagent that binds to the extracellular domain of CD28 or a fragment thereof;

[0304] ii. determining mCD28 downstream signaling in the presence of said obtained reagent; and

[0305] iii. selecting at least one agent that neither significantly agonizes nor significantly antagonizes mCD28 signaling;

[0306] Thereby, the reagent of the present invention is produced.

[0307] In some embodiments, the agent is a clearing agent. In some embodiments, the agent is used to remove sCD28 from a subject. In some embodiments, the agent specifically binds CD28. In some embodiments, the agent specifically binds sCD28.

[0308] In some embodiments, the method further comprises separating and / or extracting the reagent from the host cell. In some embodiments, the method further comprises separating and / or extracting the reagent from the culture medium of the host cell. In some embodiments, the method further comprises purifying the reagent from the host cell or the culture medium of the host cell.

[0309] In some embodiments, the method further comprises testing the obtained reagents for binding to mCD28 and selecting at least one reagent that does not bind to mCD28. In some embodiments, the method further comprises testing the obtained reagents for binding to sCD28 of the subject and selecting at least one reagent that binds to sCD28 of the subject. In some embodiments, the subject is a cancer patient. In some embodiments, the subject is an autoimmune patient.

[0310] In some embodiments, obtaining the reagent comprises immunizing an organism with a CD28 extracellular domain or a fragment thereof and collecting antibodies from the immunized organism. In some embodiments, the organism is a mouse. In some embodiments, the organism is selected from rabbits, mice, rats, sharks, camelids, chickens, goats, and bacteriophages. In some embodiments, the camelids are selected from camels and llamas. In some embodiments, collecting comprises drawing blood. In some embodiments, collecting comprises:

[0311] a. Extracting B cells from the spleen of an immunized organism;

[0312] b. fusing the extracted B cells with myeloma cells to produce hybridomas; and

[0313] c. Collecting antibodies from hybridomas.

[0314] In some embodiments, obtaining an agent includes screening an agent library that binds to the CD28 extracellular domain or a fragment thereof and selecting an agent that binds in this way. In some embodiments, the library is a phage display library. In some embodiments, the library is an immune library derived from spleen B cells. In some embodiments, the library is an IgG library. In some embodiments, the library is a Fab library. In some embodiments, the library is a library of VHH antibodies. In some embodiments, the library is a library of single-chain, single-domain or nano-antibodies. In some embodiments, obtaining an agent includes sequencing an agent. In some embodiments, obtaining an agent includes generating a recombinant form of an agent. In some embodiments, selecting an agent includes sequencing an agent. In some embodiments, selecting an agent includes generating a recombinant form of an agent. In some embodiments, the recombinant form is generated by the sequence of the agent. In some embodiments, the method further includes humanizing the agent.

[0315] The expression of nucleic acid molecules encoding agents in cells is well known to those skilled in the art. It can be carried out by a variety of methods such as transfection, viral infection, or direct modification of the cell genome. In some embodiments, the gene is in an expression vector (such as a plasmid vector or a viral vector). One such example of an expression vector containing p16-Ink4a is the mammalian expression vector pCMV p16 INK4A available from Addgene.

[0316] The vector nucleic acid sequence generally contains at least an origin of replication for propagation in cells and optionally other elements, such as heterologous polynucleotide sequences, expression control elements (e.g., promoters, enhancers), selectable markers (e.g., antibiotic resistance), polyadenine sequences.

[0317] The vector can be a DNA plasmid delivered via a non-viral method or by a viral method. The viral vector can be a retroviral vector, a herpes virus vector, an adenoviral vector, an adeno-associated virus vector or a poxvirus vector. The promoter can be active in mammalian cells. The promoter can be a viral promoter.

[0318] In some embodiments, the nucleic acid sequence encoding the agent is operably linked to a promoter. The term "operably linked" refers to a nucleotide sequence of interest that is linked to one or more regulatory elements in a manner that allows the nucleotide sequence to be expressed (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into a host cell).

[0319] In some embodiments, the vector is introduced into the cell by standard methods, including electroporation (e.g., as described in From et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985)), heat shock, infection with a viral vector, high-speed ballistic penetration by small particles having nucleic acids within or on the surface of a bead or small particle array (Klein et al., Nature 327. 70-73 (1987)), and / or the like.

[0320] The term "promoter" as used herein refers to a group of transcription control modules that are clustered around the start site of RNA polymerase (i.e., RNA polymerase II). The promoter is composed of discrete functional modules, each of which is composed of approximately 7-20 bp of DNA and contains one or more recognition sites for transcriptional activators or repressors.

[0321] In some embodiments, the nucleic acid sequence is transcribed by RNA polymerase II (RNAP II and Pol II). RNAP II is an enzyme found in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNAs and microRNAs.

[0322] 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 can be purchased from Invitrogen; pCI, which can be purchased from Promega; pMbac, pPbac, pBK-RSV and pBK-CMV, which can be purchased from Strategene; pTRES, which can be purchased from Clontech, and their derivatives.

[0323] In some embodiments, the present invention uses expression vectors containing regulatory elements from eukaryotic viruses (such as retroviruses). SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papilloma virus include pBV-1MTHA, and vectors derived from Epstein-Barr virus include pHEBO, and p2O5. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and vectors that allow protein expression under the guidance of SV-40 early promoter, SV-40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters that show effective expression in eukaryotic cells.

[0324] In some embodiments, the recombinant viral vector of the advantage such as lateral infection (lateral infection) and target specificity is provided for in vivo expression.In one embodiment, lateral infection is intrinsic in the life cycle of for example retrovirus, and is the process that single infected cell produces many progeny virions, and this progeny virion sprouts (budoff) and infects adjacent cell.In one embodiment, the result is that large tracts of land are infected rapidly, and wherein major part is not infected by original virus particle at first.In one embodiment, the viral vector that can not spread laterally is produced.In one embodiment, if the purpose of expectation is only to introduce specific gene into the target cell of local quantity, then this characteristic can be useful.

[0325] Various methods can be used to introduce the expression vector 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 transfection or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see US Pat. Nos. 5,464,764 and 5,487,992 for positive and negative selection methods.

[0326] It will be appreciated that, in addition to the necessary elements for transcription and translation of the inserted coding sequence (encoding a polypeptide), the expression constructs of the invention may also include sequences engineered to optimize the stability, production, purification, yield or activity of the expressed polypeptide.

[0327] In another aspect, there is provided an agent produced by the method of the invention.

[0328] In another aspect, a pharmaceutical composition is provided comprising an agent produced by the method of the present invention and a pharmaceutically acceptable carrier, excipient or adjuvant.

[0329] As used herein, the term "about" when combined with a value refers to plus or minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm +- 100 nm.

[0330] Note that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "polynucleotide" includes a plurality of such polynucleotides, and reference to a "polypeptide" includes one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It should also be noted that the claims may be drafted to exclude any optional elements. Thus, this statement is intended to serve as antecedent basis for the use of exclusive terminology such as "solely," "only," and the like in conjunction with a limitation of claim elements, or use of a "negative" limitation.

[0331] In those cases where a convention similar to "at least one of A, B, and C, etc." is used, such construction is generally intended to be in the sense that one skilled in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include, but is not limited to, systems having only A, only B, only C, having A and B together, having A and C together, having B and C together, and / or having A, B, and C together, etc.). One skilled in the art would further understand that, in practice, any antonymous conjunctions and / or phrases presenting two or more optional terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, any of the terms, or both of the terms. For example, the phrase "A or B" would be understood to include the possibility of "A" or "B" or "A and B."

[0332] It should be understood that certain features of the invention described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention described in the context of a single embodiment for brevity may also be provided individually or in any suitable sub-combination. All combinations of embodiments related to the invention are specifically encompassed by the invention and disclosed herein, just as each combination is individually and explicitly disclosed. In addition, all sub-combinations of various embodiments and elements thereof are also specifically encompassed by the invention and disclosed herein, just as each such sub-combination is individually and explicitly disclosed herein.

[0333] After examining the following examples, other objects, advantages and novel features of the present invention will become apparent to those skilled in the art, and these examples are not intended to be limiting. In addition, as described above and as claimed in the following claims, each of the various embodiments and aspects of the present invention is experimentally supported in the following examples.

[0334] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0335] Example

[0336] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are explained fully in the literature.See, e.g., “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, RM, ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Maryland (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (eds) “Genome Analysis: A Laboratory Manual Series”, Volumes 1-4, Cold Spring Harbor Laboratory Press, New York (1998); U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Vols. I-III Cellis, JE, ed. (1994); "Culture of Animal Cells-A Manual of Basic Technique" by Freshney, Wiley-Liss, NY (1994), 3rd edition; "Current Protocols in Immunology" Vols. I-III Coligan JE, ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization-A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.

[0337] Materials and methods

[0338] Antibody —Commercial mouse monoclonal anti-CD28 clone #CD28.2 (Biolegend, catalog number 302902) and FITC conjugated (Biolegend, catalog number 302906). Goat polyclonal anti-CD28 (R&D system, catalog number AF-342-PB). FITC conjugated anti-human PD-L1 (BD bioscience, catalog number 558065). APC conjugated anti-human PD-L2 (Biolegend, catalog number 345508). PE conjugated anti-human IDO (R&D system, catalog number IC6030P). Goat anti-mouse IgG Alexa Fluor 647 (Biolegend, catalog number 405322). Donkey anti-human IgG (H+L) Alexa Fluor 647 (Jackson immuneresearch, catalog number 709-605-149). Goat anti-mouse IgG HRP (Jackson immune research, catalog number 115-035-071). Anti-human CD3 clone OKT3 (Biolegend, catalog number 317304). Anti-human PD-1 pembrolizumab (MK-3475). Human IgG (Sigma, cat# I4506).

[0339] Production of recombinant soluble human CD28a —CD28a cDNA was subcloned into pCDNA3·1 vector, in which the c-terminal TEV protease cleavage site was before the six histidine residues to facilitate affinity purification. This plasmid was used to transfect HEK293 cells, and soluble recombinant CD28a was purified via immobilized metal affinity chromatography (IMAC). After the second IMAC, the pooled material was subjected to His-tag cleavage using TEV protease to remove the His-tag and TEV protease.

[0340] ELISA—Commercial ELISA kits were used to quantify the amount of human interferon-γ (Biolegend, catalog #430103), human interleukin 2 (Biolegend, catalog #431802), human interleukin 6 (Biolegend, catalog #430502), human interleukin 10 (Biolegend, catalog #430603), human tumor growth factor β1 (Biolegend, catalog #436708), human interleukin β1 (Biolegend, catalog #437004), and human CD28 (R&D system, catalog #DY342). Cell proliferation and survival (MTT assay) were performed according to the manufacturer's instructions (Roche, catalog #11465007001). Kynurenine (IDO activity) ELISA kit was performed according to the manufacturer's instructions (ImmuSmo catalog #BAE-2200).

[0341] Cytokine multiplex analysis - Simultaneous assessment of several cytokines was performed on the Magpix system (Millipore) using ProcartaPLex (Invitrogen, catalog number PPX-07-MXXGPY2).

[0342] Flow cytometry — Typically, cells were kept on ice during all steps. Prior to staining, 5×10 cells were blocked with 50 μg / mL human IgG (Sigma, catalog number I4506) in FACS buffer (PBS containing 0.1% BSA). 5 Cells were incubated for 15 minutes per well. Antibodies were used at the concentration recommended by the manufacturer and incubated in the dark for 30 minutes. Incubation was performed in a 96-well U-bottom plate at a volume of 100 μL. Cells were washed twice with 200 μL FACS buffer and then transferred to FACS tubes for analysis with 150 μL FACS buffer. Cells were analyzed on a Gallios flow cytometer (Beckman Coulter) using Kaluza for Gallios flow cytometry acquisition software.

[0343] Cell lines and isolation of human immune cells — Jurkat leukemic T-cell lymphoblastoid cell line clone E6.1 and SCC-25 tongue squamous cell carcinoma were obtained from ATCC. PBMCs were isolated from fresh blood samples of healthy donors using standard lymphocyte separation medium (MBP, catalog number 850494). Negative selection was performed using Rossette SEP TM Human T cell enrichment kit (STEMCELL, catalog number 15061) was used to isolate CD3 cells from fresh blood samples of healthy donors. TMHuman CD4 T Cell Enrichment Kit (STEMCELL, catalog number 19059) was used to isolate CD4 cells from fresh blood samples of healthy donors. TM The human monocyte enrichment kit (STEMCELL, catalog number 17952) was used to isolate monocytes from fresh blood samples of healthy donors. All cells were grown in complete RPMI-1640 medium supplemented with 10% HI-FCS and a penicillin / streptomycin mixture.

[0344] Dendritic cell 1×10 6 Monocytes were cultured at a density of 1000 μg / mL and the culture medium was refreshed on days 3 and 6. Immature dendritic cells (iDC) were induced for 6 days with 50 ng / mL GM-CSF and 20 ng / mL IL-4. When necessary, iDC were further differentiated into mature dendritic cells for 48 hours by adding 100 ng / mL LPS. The resulting cell populations were tested for their indicated phenotypes by FACS analysis of relevant markers and secretion analysis of characteristic cytokines.

[0345] Protease inhibitors - At the beginning of each experiment, protease inhibitors were added at the indicated concentrations. In a one-week trial, another portion of inhibitors was added at final concentrations after 3 days. The protease inhibitors used were TAPI-1 (Cayman, catalog number 18505), GM6001 (Santa Cruz, catalog number SC-203979), TMI-1 (Sigma, catalog number PZ0336) and GI254023X (Sigma, catalog number SML0789). Where mentioned, the protease mixture consisted of a mixture of equimolar ratios of TAPI-1 and GM6001.

[0346] PHA activates CD4 T cells or Jurkat T cell lines to produce soluble CD28 —1×10 5 Jurkat cells or CD4 T cells were incubated with the indicated concentrations of phytohemagglutinin (Sigma, catalog number L8902) and various protease inhibitors for an additional 5 days (Jurkat) or 7 days (CD4 T cells).

[0347] SEB or CMV activates PBMCs to produce soluble CD2 8—0.3×10 cells were stimulated with 0.5 ng / mL SEB (Sigma, catalog number S4881) in a 48-well plate at 37°C in the presence or absence of various protease inhibitors at indicated concentrations. 6 Alternatively, in a 96-well plate format, 0.1 × 10 PBMCs were stimulated with 0.5 ng / mL SEB. 6For CMV stimulation, 0.5 × 10 PBMCs were stimulated with 0.5 μg / mL CMV peptivator (Milteny Biotec, catalog number 130-093-435) in a 96-well plate at 37°C in the presence or absence of various protease inhibitors at the indicated concentrations. 6 PBMCs were cultured for 2-5 days. For continuous shedding experiments, PBMCs were stimulated with SEB or CMV for 24 hours in 24-well plates, the cells were removed and washed 3 times with RPMI without stimulants, and plated again in 96-well plates. Samples were taken at the indicated times and placed in frozen conditions until soluble CD28 was examined.

[0348] Mixed lymphocyte reaction —1×10 5 Immature DCs and 5×10 5 Spontaneous CD3 T cells isolated from the culture medium were mixed for 6 days.

[0349] SEB or CMV stimulation assays with ectopic recombinant human CD28, human CTLA-4, and human CD80 For CMV stimulation, 0.5 × 10 cells were stimulated with 0.5 μg / mL CMV peptivator (Milteny Biotec, catalog number 130-093-435) in a 96-well plate at 37°C in the presence or absence of indicated concentrations of recombinant human CD28 (R&D systems, catalog number 342-CD), human CTLA-4 (R&D systems, catalog number 434-CT), and human CD80 (R&D systems, catalog number 140-B1). 6 PBMCs (from healthy donors or cancer patient donors) were cultured for 2-5 days. For SEB, 1×10 5 PBMCs were cultured for 72 hours. When indicated, anti-PD-1 or human IgG was added at a final concentration of 5 μg / mL.

[0350] Autologous monocyte CD3 MLR —0.5×10 6 T cells from the same CMV-reactive donor were compared with 0.5 × 10 5 Monocytes were mixed and stimulated with 0.5 μg / mL CMV peptivator at 37°C for 6 days in the presence or absence of indicated concentrations of treatment.

[0351] Stimulation of monocytes with recombinant human CD28 — In the presence of the indicated concentrations of recombinant human CD28, 1.5 × 10 6The mononuclear cells were plated in RPMI medium containing 100-100U / ml IFNγ (R&D system, catalog number 285-IF) in a 24-well plate for 48 hours. The indicated phenotype of the generated cell population was tested by FACS analysis of relevant markers (IDO, PD-L1 and PD-L2) and secretion analysis of characteristic cytokines (IL-6).

[0352] Stimulation of T cells with OKT3 — 0.1×10 cells were stimulated with the indicated amount of anti-CD3 clone OKT3 at 37°C. 6 Isolated CD3 T cells (from healthy donors) were added for 48-72 hours. When stated, recombinant human CD80-Fc (2 μg / mL, R&D system) was added in soluble form. Antibodies against CD28 or controls were added in soluble form at the indicated concentrations.

[0353] CD86 blocking FACS — At room temperature, 0.5×10 cells stably transfected with human CD28 were cultured in the absence or presence of anti-CD28 antibody (20 μg / mL). 6 HEK293 cells were incubated with 5 μg / mL CD86-Fc (R&D systems, catalog number 141-B2) for 30 minutes. The cells were washed and removed for a second binding on ice for 20 minutes using anti-human heavy and light chain antibodies conjugated to fluorophores at a dilution of 1:5000.

[0354] Co-culture of the SCC-25 cancer cell line with monocytes in a trans-well assay —4 × 10 4 SCC-25 cells were plated on the bottom of a 24-well plate, with 1 × 10 5 Monocytes were placed in cell culture inserts (Millipore, catalog number MCHT241148) for 4 days.

[0355] Identification, cloning and sequencing of CD28 mRNA variants- Human PBMCs were stimulated with 0.5 ng / mL SEB (Sigma, catalog number S4881) for 7 days at 37°C. Human CD4 T cells were incubated with phytohemagglutinin (2 μg / mL, Sigma, catalog number L8902) for 7 days at 37°C. Total RNA was extracted from pellets of activated and immature immune cells using the Qiacube automation system (Qiagen) using the RNeasy mini kit (Qiagen, catalog number 74106). 500 ng RNA was taken and RT was performed using a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific, catalog number 4374966). The negative control for the RT reaction was a tube without RNA and another tube without reverse transcriptase. Take 1 μ L cDNA and use the following forward primer (CD28F) 5'-ATGCTGAGGCTGCTCTTGGCTCTCAAC-3' (SEQ ID NO: 38) and reverse primer (CD28R) 5'-TCAGGAGCGATAGGCTGCGAAGTCGCG-3' (SEQ ID NO: 39) to perform PCR. The PCR product is loaded on a 1% agarose gel. The PCR product is cut from the gel and extracted using a QIAquick gel extraction kit. Sanger sequencing is performed using the CD28F primer indication.

[0356] Detection of soluble human CD28 in plasma of cancer patients - Twenty frozen plasma samples from each of 10 different cancer indications and healthy donors were purchased from DxBiosamples (San Diego, CA, USA). Plasma samples were diluted 1:20 and analyzed for soluble human CD28 by ELISA. Samples with high sCD28 were analyzed again at appropriate dilutions.

[0357] Direct CD28 EIA— Unless otherwise discussed, screening was performed using Corning high binding plates or equivalent. Each well was coated with 200-300 ng of human CD28-Ig chimera (R&D, catalog number 342-CD), mouse CD28-Ig chimera (R&D, catalog number 483-CD), or BSA-conjugated dimeric peptide consisting of the amino acid sequence of the CD28 stem region (Gly137-Pro152). The plates were blocked with 5% milk or 1% casein in PBS for 1 hour at room temperature (RT). The plates were washed 3 times with PBST and incubated with the antibodies under study after detection with specific goat anti-mouse HRP Fc diluted 1:5000. Positive controls were mouse anti-human CD28 clone 28.2 or mouse serum from immunized mice. Hybridoma supernatant cultures were screened without dilution.

[0358] Simulating the elimination of soluble CD28 in the plasma of cancer patients . Antibodies or recombinant proteins were coated on tosyl-activated magnetic beads (Invitrogen, catalog number DY-14203) as described by the manufacturer's protocol. The beads were removed to display the indicated amount of antibody and mixed with plasma samples from cancer patients. The mixture was incubated for 2 hours at 1000 RPM at 37° C. in a thermomixer, then the beads were removed using a DynaMag magnet (Invitrogen, catalog number 12321D), and the samples were examined using a CD28-specific ELISA.

[0359] Antibody Sequencing . The antibodies were provided to Rapid Novor for amino acid sequencing. Sequencing was performed using standard methods, which briefly included: LC-MS analysis after enzymatic digestion with six enzymes (pepsin, trypsin, chymotrypsin, elastase, LysC and Asp N). Digestion was performed with disulfide reduction and alkylation. LC-MS / MS analysis was performed using a Thermo-Fisher Q-exactive mass spectrometer. In both the heavy and light chains of each antibody, 100% of the amino acid residues were covered by at least 5 peptide scans with significant supporting fragment ions. CDRs were determined using the Chothia protocol.

[0360] Example 1: Human CD28 undergoes proteolytic shedding during chronic stimulation

[0361] Soluble CD28 (sCD28) was detected by ELISA in chronically stimulated human PBMC cultures ( Figure 1). This phenomenon was evident regardless of the nature of the stimulus, artificial (SEB) or physiological (CMV), indicating its stability. The origin of soluble CD28 is shedding from membrane forms, since treatment with TAPI-1 and GM6001 (a broad MMP and ADAM17 inhibitor) reduced the amount of sCD28 in a dose-dependent manner ( Figure 1 ). The cell source of shed CD28 is T cells, such as Figure 2 Chronic stimulation of Jurkat T cell lines or human CD4 T cells from peripheral blood of healthy donors with PHA resulted in the production of sCD28 ( Figure 2 , upper panel). Treatment with TAPI-1 and GM6001 reduced the amount of sCD28 at each PHA concentration ( Figure 2 , upper figure), while at a fixed PHA concentration, it was dose-dependent ( Figure 2 , figure below).

[0362] Treatment with the highly specific ADAM-10 inhibitor GI254023X resulted in almost complete inhibition of sCD28 release from activated immune cells in a dose-dependent manner ( Figure 3A , lower figure). Similar results were observed with the ADAM-17-specific inhibitor TMI-1 ( Figure 3B , lower figure). The survival of immune cells was monitored by MTT assay, examining the metabolic activity of cells in culture. The results showed no significant differences between treatment with and without either ADAM inhibitor, suggesting that low sCD28 levels were caused by blockade of protease activity rather than an artifact of cell death caused by protease inhibitors ( Figure 3A -B, above).

[0363] The generation of sCD28 has also been validated in more physiological systems. First, using isolated autologous dendritic cells and CD4 T cells that mimic the physiological stimulation of T cells by antigen presenting cells. When the two cell populations were mixed, the increase in sCD28 was obvious, and when CMV was added to the culture, the increase in sCD28 became even more significant ( Figure 4A ). This shows that when chronic stimulation occurs, human CD28 protein undergoes proteolytic shedding.

[0364] Next, CMV peptide ( Figure 4B ) or SEB( Figure 4C ) were used to stimulate human PBMCs for 24 hours. Afterwards, the cells were washed to remove the stimulant and plated again at various time periods without any stimulatory signal. After this, the culture medium was checked for the presence of sCD28. Over time, the accumulation of sCD28 was clearly visible. In addition, Figure 4DAs shown, accumulation depends on the activity of ADAM-10 and ADAM-17. After SEB stimulation, the addition of different concentrations of specific inhibitors led to a decrease in the amount of sCD28 quantified after 120 hours. This study could explain the presence of a large amount of soluble CD28 in the blood of patients, since CD28 shedding occurs after primary activation of T cells and does not necessarily require constant or repeated stimulation.

[0365] Final evidence that the origin of sCD28 is proteolytic shedding was obtained by observing that a known alternative splice variant of CD28 is significantly downregulated in activated lymphocytes ( Figure 5 ). Four CD28 mRNA products were detectable in unstimulated samples, whereas only two were found in activated cells. Sanger sequencing confirmed that the top band corresponds to the full-length mature CD28 that is membrane-bound upon translation, while the second band (black arrow, ~500 bp) has previously been shown to be an alternatively spliced ​​product that results in secreted truncated protein. Sanger sequencing was used to confirm that this band from the naive sequence is a splice variant lacking exon 3 and, therefore, the transmembrane domain. SEB or PHA stimulation of PBMCs and T cells results in preferential expression of full-length CD28 mRNA, accompanied by suppression of spliced ​​transcripts ( Figure 5 , lanes 5 and 7). Together, these results demonstrate that the origin of sCD28 in activated T cells is proteolytic shedding rather than alternative splicing at the gene level.

[0366] Example 2: Soluble human CD28 has immunosuppressive activity

[0367] from Figure 1 As can be seen in the Figure 2, reducing the levels of sCD28 using a protease inhibitor cocktail directly correlated with an increase in T cell activation, as indicated by the levels of secreted IFNγ, indicating that sCD28 has an immunosuppressive function. Increasing concentrations of the protease inhibitor cocktail resulted in reduced levels of sCD28 in the cell culture medium, and these lower levels of sCD28 were negatively correlated with higher levels of secreted IFNγ. To further explore the immunosuppressive effects of sCD28, recombinant human CD28 lacking the transmembrane and cytoplasmic domains was added to human PBMC cultures stimulated with CMV. This resulted in a dose-dependent inhibition of IFNγ secretion ( Figure 6 This immunosuppressive effect was observed in different human PBMC donors, confirming the stability of this signaling axis blocked by sCD28.

[0368] In parallel, interleukin-6 secretion ( Figure 7 and 8A ) and interleukin-10 ( Fig. 8A) were evident. These cytokines reportedly exhibited suppression of immune effector activity (IL-10) and a tilt of the immune system toward a type 2 immune response that can support cancer proliferation and angiogenesis through STAT-3 signaling (IL-6). In addition, comparisons were made with soluble CTLA-4 (mimicking Abatacept, a registered therapy for autoimmune disorders) and revealed that the overall effects on the immune system were similar in terms of cytokine secretion profiles ( Fig. 8A ).

[0369] 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. S3Live-Cell monitors lymphocyte aggregation, a hallmark of immune activation, and takes pictures every 12 hours. Figure 8C As can be seen in the results, SEB had little effect on lymphocytes in the presence of recombinant human sCD28. It is well established that antigen presenting cells (APCs) aggregate with each other and with other cell types during in vitro immune responses, and that aggregation is essential for antigen-specific activation of dormant lymphocytes. Soluble CD28 appears to reduce the number and size of clusters formed during SEB immune responses, meaning that it inhibits the first step in T cell-specific activation of APCs.

[0370] 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 and 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 8B ).

[0371] sCD28 has a similar immunosuppressive effect on monocytes. Indoleamine 2,3-dioxygenase (IDO) is involved in immune regulation through its ability to catabolize the essential amino acid tryptophan. It is expressed by different immune cells and also by a variety of cancer cells. Tryptophan shortage inhibits the maturation and proliferation of T lymphocytes, while kynurenine, the final product of tryptophan catabolism, is also known as an immunosuppressive metabolite, which promotes immune tolerance under various physiological and pathophysiological conditions. To test the effect of sCD28 on IDO, isolated human monocytes were stimulated with IFNγ (1000U / mL) in the presence of control human IgG or recombinant human CD28 (10μg / mL) for 48 hours. After incubation, monocytes were intracellularly stained for human IDO ( Fig. 8ETo facilitate intracellular staining, cells were fixed and permeabilized with the BD Cytofix / Cytoperm buffer kit. IDO activity of the culture media from different treatments was assessed using the ImmuSmol specific kynurenine ELISA kit ( Fig.8D ). sCD28 strongly enhanced the expression of IDO in monocytes.

[0372] Furthermore, sCD28 was surprisingly found to be a potent inhibitor of anti-PD1 immunotherapy. MK-3475 (pembrolizumab or Keytruda, Merck) is an approved drug with unprecedented efficacy in multiple cancer indications. Its addition to PMBC cultures increased the secretion of pro-inflammatory cytokines (IFNγ and IL-2), but the presence of sCD28 completely abolished this immune activation effect ( Fig. 9A ).

[0373] Similar results were again observed in the context of the MLR. The MLR was run as before only in the presence and absence of sCD28 and in the presence and absence of anti-PD1 antibody (MK3475, 5 μg / mL). Fig. 9B ). As expected, MK-3475 increased the secretion of IFNγ and decreased the secretion of TGFβ. Notably, the effect of MK-3475 was significantly reduced in the presence of sCD28.

[0374] 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 in the presence of sCD28 was examined. Isolated human monocytes were stimulated with IFNγ (1000 U / mL) for 48 h 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 ( Fig. 9C , left) and PD-L2( Fig. 9C , right). Both ligands are upregulated on monocytes cultured with sCD28, suggesting a possible way in which sCD28 may circumvent the effects of anti-PD-1 immunotherapy.

[0375] Example 3: Discovery of soluble human CD28 in the plasma of cancer patients

[0376] sCD28 levels in cancer have only been shown in a few breast cancer patients, and the levels were found to be only slightly above those observed in healthy individuals (Isitmangil, G., In vivo, 2016). Although the authors suggested that sCD28 could potentially be used as a marker for breast cancer, no functional link was proposed. Now that it is known that soluble CD28 can actually enhance cancer evasion of the immune system, a survey of 220 samples was conducted covering 10 different cancer indications and 20 samples from healthy donors. The survey found elevated levels of sCD28 in several cancers, sometimes several orders of magnitude higher than those seen in healthy controls and even breast cancer patients ( Fig. 10A ). In fact, compared to the levels of sCD28 found in certain melanoma, colorectal cancer, ovarian cancer, NSCLC, and head and neck cancer patients, the levels in breast cancer patients appear to be comparable to healthy individuals. This investigation was conducted using antibodies #1 and #3 of the present invention. Surprisingly, such high levels have not been reported previously in any study known to the inventors. Therefore, 20 melanoma patient samples were tested by sandwich ELISA using the antibodies of the present invention (antibody #3 for capture and antibody #1 for detection) and also a commercially available CD28 kit purchased from R&D Systems (category #DY342). Although the antibodies of the present invention detected very high levels of sCD28 in three samples and lower levels in another 14 samples, the commercially available kit did not detect sCD28 in any of the samples ( Fig. 10B ). Similar results were found with the commercially available CD28 ELISA kit BMS290 (available from Thermo Fischer Scientific), which has been used in the studies of Isitmangil et al. on breast cancer. The commercially available kit cannot detect sCD28 shed from human cells, which could explain why high sCD28 in cancer patients has been unknown until now.

[0377] To further elucidate the role of sCD28 in cancer, PBMCs were isolated from cancer patients with different indications. The cells were stimulated for 3 days with SEB (5 ng / mL) alone, with MK-3475, with recombinant human sCD28, or with a combination of both molecules. In the presence of sCD28, the concentration of human IFNγ in the cell supernatant from all donors was greatly reduced ( 1.5 ng / mL) even in the presence of MK-3475. Fig. 10C ). In fact, sCD28 negated the effect of MK-3475.

[0378] Next, cells of the head and neck cancer cell line SCC-25 were incubated alone or with monocytes in a well migration assay. SCC-25 cells grown alone were given IL-6 as a positive control, and the ( Fig. 10D , top) and measured as % confluency ( Fig. 10D , below) cell proliferation did increase. Growing cancer cells in the presence of monocytes also increased proliferation, but by far the greatest increase was observed when the co-culture included sCD28. This data further supports that sCD28 has a pro-cancer role.

[0379] Example 4: sCD28 inhibits CD80-Fc efficacy

[0380] CD80 is one of the two major ligands for mCD28, along with CD86. The extracellular domain of CD80 fused to the Fc portion is used as an immunostimulatory molecule and is under investigation as a cancer therapy. To examine the effect of sCD28 on the efficacy of CD80-Fc, isolated CD3 human T cells were stimulated with plate-bound anti-CD3 antibody (OKT3, 2 μg / mL) in the presence of 2 μg / mL soluble recombinant human CD80-Fc. As expected, CD80-Fc increased IFNγ secretion. However, the addition of sCD28 counteracted the secondary activation effect of CD80-Fc ( Fig.11A Similarly, when isolated PBMCs were stimulated with CMV peptide for 3 days and then incubated with sCD28, the amount of CD80-Fc needed to be increased to generate the expected immune response ( Fig. 11B ).

[0381] Example 5: Effects of sCD28 on cancer in vivo

[0382] Since mice do not splice mCD28, the role of sCD28 cannot be easily examined in mouse models. The closest option is to give mice recombinant sCD28 to mimic a situation where sCD28 levels are elevated. This has been studied in the H22 syngeneic mouse model. Balb / c fully immunocompetent mice received allogeneic transplants of H22 hepatocellular carcinoma. These cells grew even in fully immunocompetent mice, and the addition of anti-PD-1 therapy almost completely abolished tumor growth ( Fig. 12A When recombinant human sCD28 was added, the effects of anti-PD-1 therapy were almost completely abolished in two of the mice ( Fig. 12B ). This suggests that in certain subjects, elevated sCD28 levels can have a highly deleterious effect on cancer progression.

[0383] Example 6: Characterization of antibody-based agents that abolish the immunosuppressive effects of sCD28 (shear resistance)

[0384] The discovery that human CD28 undergoes proteolytic processes of ADAM10 and ADAM 17 has prompted the examination of candidate regions of its polypeptide sequence, which show potential sensitivity to proteolytic release. Studies have shown that ADAM 10 and ADAM17 prefer leucine, valine and aromatic residues at the P1' site. The most attractive sequence region in human CD28 is the stem segment, ranging from histidine 134 to proline 152 (SEQ ID 10: HVKGKHLCPSPLFPGPSKP), connecting the globular IgV domain to the transmembrane region. This region maintains 3 total leucine and valine residues, as well as phenylalanine residues, and it is predicted that this region does not contain any secondary structural elements that may hinder the entry of proteases. It is worth noting that the stem region also contains cysteine ​​141, which forms a disulfide bond that promotes the homodimerization of CD28. In order to produce antibodies or antibody fragments that specifically bind to CD28 stem region and potentially block different proteases from entering the CD28 that comes off, while avoiding any damage to CD28 oligomer structure and function, CD1 mice are immunized with dimer peptides that simulate CD28 stem region. The peptide sequence used for immunity is SEQ ID:40, GKHLCPSPLFPGPSKPK, and the addition of C-terminal lysine is to have free amino groups to allow the use of hydrazide chemistry to carry out KLH or BSA conjugation. Conjugation is carried out between hydrazide-terminated CD28 peptides and S-4FB-modified BSA, and the latter produces free aldehydes to carry out site-specific conjugation. Dimerization is confirmed by running the peptide on a non-denaturing gel.

[0385] Six mice were immunized with BSA-conjugated peptides. Mouse tail-bleed sera were routinely examined by ELISA for binding to target antigens, recombinant human CD28 (rhCD28) and mixed peptides conjugated to KLH.

[0386] The high-throughput screening platform was used for initial clone screening for binding to the antigen target in ELISA. Using culture supernatant as the primary antibody and developing with HRP-conjugated goat anti-mouse IgG (γ) and TMB, approximately 2000 hybridomas were generated and screened in the initial EIA for rhCD28. Twenty clones were obtained, all of which were IgG2a antibodies, and were further expanded for purification and typing. ELISA experiments confirmed that all 20 IgGs had various levels of significant binding to recombinant soluble CD28 (Table 1).

[0387] Table 1: Anti-chopping antibodies

[0388]

[0389]

[0390] The three highest binding clones were sequenced and found to be the same antibody (hereafter referred to as clone M9), so experiments were continued with this antibody. The sequence of the M9 antibody can be found above. Serial dilutions of antibody M9 were used to confirm its specific binding to recombinant human sCD28 and to the stalk region peptide ( Fig.13A Interestingly, although the antibody detected recombinant human sCD28, it failed to detect sCD28 that was actually shed from immune cells ( Fig. 13B ). This strongly suggests that the antibody binds at the cleavage site and that the deisotope it binds to is incomplete in the cleaved form. Direct inhibition of cleavage was examined by mixing rhCD28 with ADAM 10 or ADAM17 in the presence and absence of antibody M9. The resulting rhCD28 peptides were sequenced by mass spectrometry to determine whether cleavage occurred.

[0391] These results suggest that binding to the stalk region may not have a direct effect on mCD28 signaling. However, the monoclonal antibody MAB342, clone 37407 from R&D Systems, has been reported to bind in the stalk region of CD28 (see International Patent Application WO2004096139). In addition, R&D Systems reported that this antibody is a CD28 agonist (rndsystems.com / products / human-cd28-mab-clone-37407-37407_mab342). Therefore, the binding of this antibody was studied. MAB342 was found to bind to recombinant human CD28-Fc as well as recombinant human CD28a (a soluble splice variant lacking the stalk region sequence), but not to recombinant mouse CD28 ( Fig. 13C However, when tested for binding to the human CD28 stalk peptide, no binding was observed, strongly suggesting that this antibody does not actually bind to the CD28 stalk region ( Fig. 13C ).

[0392] Example 7: Characterization of Antibody-Based Agents to Eliminate the Immunosuppressive Effects of sCD28 (Clearance)

[0393] Three antibodies that bind strongly to sCD28 were obtained and investigated for their potential utility as clearing agents to reduce sCD28 levels. The sequences of the three antibodies are provided above.

[0394] To confirm the ability of these three antibodies to bind to human sCD28, human CD3 T cells were stimulated with PHA, and sCD28 was measured by sandwich ELISA using two antibodies of the present invention. Representative results using antibody 1 as the detection antibody and antibody 2 as the capture antibody are shown in Fig.14ASimilarly, human PBMCs were stimulated with SEB, and the antibodies of the invention were again able to robustly detect sCD28 ( Fig. 14B ). When the protease inhibitors TAPI, GI254023X, and TMI-1 were used, lower levels of sCD28 were detected, confirming that sCD28 was indeed detected.

[0395] Antibodies #1 and #2 were compared to a control commercial CD28 antibody, CD28.2, known to bind mCD28. Direct ELISAs were performed with each antibody (and with mIgG as a negative control) in the presence of varying concentrations of sCD28. Fig. 14C ). Recombinant human sCD28 protein was immobilized on maxisorp ELISA plates and binding was assessed by detecting bound antibody with donkey anti-mouse IgG (H&L)-HRP and developing with TMB. Although the commercial antibody CD28.2 did bind sCD28, the binding was poor and there was only a small increase in OD even with a ten-fold increase in input. In contrast, both antibodies #1 and #2 showed stronger binding to sCD28, with antibody #1 showing nearly ten-fold higher detection and antibody #2 showing a linear relationship with increasing antibody concentration over the entire range examined. Fig.14D Direct ELISAs for each antibody are provided separately in . Interestingly, antibody #2 was also able to bind mouse CD28, while antibodies #1 and #3 were not ( Fig.14E ).

[0396] CD28.2 is known to stimulate T cell proliferation and cytokine secretion, so it acts as an agonist for mCD28. Indeed, when CD86 binding to mCD28 was measured by FACS, the addition of CD28.2 greatly reduced CD86 binding ( Fig.15A ), indicating that CD28.2 binds to or blocks the ligand binding domain of mCD28. In contrast, antibody #1 ( Fig. 15B )、antibody #2( Fig. 15C ) or antibody #3( Fig.15D ) did not block CD86 binding to mCD28, and in fact both antibodies appeared comparable to the mIgG control ( Fig.15E ).

[0397] Interferon gamma (IFNγ) secretion was measured as a proxy for pro-inflammatory cytokine secretion by T cells. In the presence of anti-CD3 stimulation, antibody CD28.2 induced robust IFNγ secretion, indicating that T cells were activated. In contrast, antibodies #1, #2, and #3 had no effect on IFNγ secretion at various concentrations ( Fig.15F). Thus, while CD28.2 acts as an mCD28 agonist, antibodies #1-3 are not agonists. Similar results were found when human PBMCs were stimulated with SEB ( Figure 15G ).

[0398] Similarly, when human isolated T cells were stimulated with anti-CD3 antibodies, CD80-Fc behaved as an agonist, increasing the secretion of IFNγ. Adding an antagonist should reduce the effect of CD80, however, when antibodies #1-3 were added, no reduction in secretion was observed ( Fig.15H ). This indicates that antibodies #1-3 are also non-antagonistic.

[0399] Finally, the ability of antibodies #1-3 to bind fully to mCD28 was examined. Although it is not necessary for the sCD28 antibody not to bind to mCD28, it is advantageous. Such an antibody can be used to specifically detect soluble proteins and will have no effect on membrane form if used therapeutically. Naive isolated CD3 T cells were evaluated by FAC to determine mCD28 binding. Cells were incubated with antibodies #1-3 (20 μg / mL dark gray histograms) or an isotype control (mIgG, 20 μg / mL light gray histograms). Binding was detected with an Alexa Fluor 647-conjugated goat anti-mouse secondary antibody. Fig.15I As can be seen, only antibody #1 fully binds to mCD28. Antibodies #2 and #3 do not bind CD28 on the cell surface and are therefore completely specific for sCD28.

[0400] Example 8: Antibodies can be used to remove sCD28 from plasma

[0401] Different amounts (0.125-2 μg) of antibodies #1 and #2 were loaded onto tosyl-activated magnetic beads according to the manufacturer's protocol (Thermo Fisher Scientific). The loaded beads were then added to plasma samples from cancer patients and the mixture was incubated in a thermomixer at 37°C at 1000 RPM for 2 hours. The beads were separated and removed from the plasma with a magnet, and the amount of sCD28 remaining in the serum was measured by ELISA. Antibody #1 ( Fig.16A ) and antibody #2( Fig. 16B ), showing that the antibodies of the present invention are able to bind to sCD28 and remove sCD28 from plasma.

[0402] Antibody #1 was further tested on various cancer samples (melanoma, colorectal cancer, and ovarian cancer) and compared to three known CD28 ligands, the B7 molecule. Approximately 1.5 μg of antibody #1, CD80, CD86, and ICOSL were each loaded onto tosyl-activated magnetic beads, and the beads were mixed with plasma as previously described. Colorectal samples ( Fig. 16C ), and two melanoma samples ( Fig.16D -E), ovarian samples ( Fig.16F ) and samples from healthy donors ( Figure 16G ) were tested in all 12 samples. Antibody #1 outperformed the other three molecules tested and in all cases was able to reduce sCD28 levels in plasma to or below the levels observed in healthy samples. In fact, in all but one of the melanoma samples ( Fig.16D ), Antibody #1 reduced sCD28 to nearly undetectable levels in all samples except for 547 (A), but even in this sample a reduction of greater than 50% was observed.

[0403] Although the present invention has been described in conjunction with specific embodiments of the present invention, it is apparent that many alternatives, modifications and variations will be apparent to those skilled in the art. Therefore, it is intended to encompass all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

1. A method for producing an agent that binds to membrane CD28 (mCD28) and inhibits protease cleavage of said mCD28, the method comprising: Obtaining an agent that binds to the extracellular domain of CD28 or a fragment thereof, testing the ability of the agent to block the cleavage of mCD28 by a protease, and selecting at least one agent that blocks the cleavage of mCD28 by the protease; or Cultivating a host cell containing one or more vectors comprising a nucleic acid sequence encoding an agent, wherein the nucleic acid sequence is a nucleic acid sequence of an agent selected by: i. obtaining a reagent that binds to the extracellular domain of CD28 or a fragment thereof; ii. testing the ability of the agent to block cleavage of mCD28 by a protease; and iii. selecting at least one agent that blocks the cleavage of mCD28 by the protease; Thereby generating an agent that binds to mCD28 and inhibits proteolytic cleavage of said mCD28.

2. The method of claim 1, wherein the protease is selected from the group consisting of ADAM 10 and ADAM 17.

3. The method according to claim 1 or 2, wherein the reagent that specifically binds to the extracellular domain of CD28 or a fragment thereof is a reagent that specifically binds to the stalk domain of CD28.

4. The method of claim 3, wherein the stem region comprises the amino acid sequence GKHLCPSPLFPGPSKP (SEQ ID NO: 9) or KGKHLCPSPLFPGPS (SEQ ID NO: 36).

5. The method of claim 3, wherein the stem region consists of the amino acid sequence HVKGKHLCPSPLFPGPSKP (SEQ ID NO: 10).

6. The method of claim 1 or 2, further comprising measuring mCD28 downstream signaling in the presence of the obtained agent, and selecting at least one agent that neither substantially agonizes nor substantially antagonizes mCD28 signaling.

7. A method for producing an agent that binds to soluble CD28 (sCD28) and is neither a CD28 agonist nor a CD28 antagonist, the method comprising: Obtaining an agent that binds to the CD28 extracellular domain or a fragment thereof, measuring mCD28 downstream signaling in the presence of the obtained agent, and selecting at least one agent that neither substantially agonizes nor substantially antagonizes mCD28 signaling; or Cultivating a host cell containing one or more vectors comprising a nucleic acid sequence encoding an agent, wherein the nucleic acid sequence is a nucleic acid sequence of an agent selected by: i. obtaining a reagent that binds to the extracellular domain of CD28 or a fragment thereof; ii. determining mCD28 downstream signaling in the presence of the obtained reagent; and iii. selecting at least one agent that neither significantly agonizes nor significantly antagonizes mCD28 signaling; This results in an agent that binds sCD28 and is neither a CD28 agonist nor a CD28 antagonist.

8. The method of claim 7, further comprising testing the obtained agents for binding to mCD28, and selecting at least one agent that does not bind to mCD28.

9. The method of claim 7 or 8, further comprising testing the obtained agents for binding to sCD28 from a cancer patient, and selecting at least one agent that binds to the sCD28 from a cancer patient.

10. The method of any one of claims 1, 2, 7 and 8, wherein said obtaining said agent comprises screening a library of agents that bind to the CD28 extracellular domain or a fragment thereof, and selecting a binding agent.

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