Methods and compositions for reducing soluble immune receptor CD28

Abstract

Methods for treating cancer and improving immunotherapy are provided, including reducing soluble immunoreceptor levels. Agents that bind to membrane immunoreceptors and inhibit proteolytic cleavage of the receptor, and agents that bind to soluble immunoreceptors and are not receptor agonists or antagonists, are also provided, as are methods for producing these agents. [Selected figure] Figure 10A

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 643,334, filed Mar. 15, 2018; U.S. Provisional Patent Application No. 62 / 643,355, filed Mar. 15, 2018; and U.S. Provisional Patent Application No. 62 / 774,254, filed Dec. 2, 2018, the contents of all of which are hereby incorporated by reference in their entirety.

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

Background Art

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

[0004] In contrast to the activating costimulatory molecule CD28, the structural homolog cytotoxic T lymphocyte-associated 4 (CTLA-4) is an inhibitory costimulatory receptor with membrane expression driven by the trigger of CD28. Both CTLA-4 and CD28 are type I transmembrane proteins. Their extracellular portions are composed of one V-set immunoglobulin superfamily (Ig-V) domain homocovalently linked by cysteine residues located outside the IgV domain proximal to the transmembrane region. CTLA-4 and CD28 are similar but differ in terms of affinity and quaternary structure arrangement. CTLA-4 has a higher binding affinity for B7 molecules, has a different dimerization mode from CD28, and has been found to have a different stoichiometric binding with the shared ligand. CD28 exhibits a monovalent binding stoichiometry, while CTLA-4 interacts in a bivalent manner. Thus, CTLA-4 binds to B7 molecules with much higher affinity and avidity than CD28, resulting in downregulation of the T cell response and being favorable for the initiation of antigen-specific tolerance.

[0005] Several costimulatory molecules have been shown to have several physiological forms. In addition to the membrane-bound form, a soluble form expressed in naive immune cells has also been reported, increasing the complexity of T cell biology. The soluble form of CD28 (sCD28) is thought to result from a selectively spliced gene product. As a result of the splicing event, a frameshift occurs and two glutamate residues are added after glycine at position 137 before translation stops. The final product lacks the entire transmembrane and cytoplasmic regions, and importantly, the cysteine residue at position 141 that mediates the disulfide bond of dimeric CD28 is missing (Magistrelli G., Biochem Biophy Res Commun, 1999). The biological function and counter-receptor binding of monomeric CD28 soluble form have been investigated (Hebbar, M., Clin Exp Immunol, 2004), and it has also been shown to inhibit T cell proliferation. Nevertheless, in the case of dimeric sCD28, it has been suggested to have a regulatory role in suppressing T cell function by binding to B7 molecules (Sun, Z., Centr Eur J Immunol, 2014; Hebbar, M., Clin Exp Immunol, 2004). Surprisingly, an increase in the number of sCD28 molecules in the sera of patients with autoimmune diseases has been reported (Wong, C.K., Rheumatol, 2005; Hamzaoui, K., Clin Exp Rheumatol, 2005; Hebbar, M., Clin Exp Immunol, 2004; Sun, Z., Clin Immunol, 2014). The exact source of sCD28 has been a subject of debate. Using an in vitro model of T cell activation that reflects the persistent inflammatory state of T cells in autoimmune patients, it has been shown that alternative soluble form transcription is suppressed during the process of T cell activation and only the full-length membrane form of CD28 is evident, while the amount of sCD28 in culture increases (Hebbar, M., Clin Exp Immunol, 2004). This phenomenon has led to the proposal that the active shedding of the membrane form of CD28 is responsible for the increase in soluble molecules in the serum, but this has not yet been proven.Rapid shedding during T cell activation has been previously described as a regulatory mechanism counteracting the continuous activation by proteolysis of adhesion molecules.

[0006] CTLA-4 limits the amplitude of the initial T cell response, while another inhibitory receptor, PD-1, suppresses peripheral T cell function. The expression of PD-1 increases during T cell activation, and its known ligands are B7 family homologs: B7-H1 (PD-L1) and B7-H2 (PD-L2). These homologs are found on APCs and cancer cells, rendering activated T cells in a state of cellular anergy and weakening the immune response. Therefore, targeted therapies against the CTLA-4 and PD-1 / PD-L1 axes have shown clinical activity in various types of cancer. Recent studies have shown that the signaling pathway of CD28 is targeted and inhibited by PD-1 (Hui, E., Science, 2017), and at the same time, it has been shown that an intact and active CD28 / B7 axis is essential for effective PD-1 therapy (Kamphorst, A.O., Science, 2017).

[0007] However, not all patients respond to PD-1-based immunotherapy or general immunotherapy, and relapses often occur. Therefore, there is a great need for ways and molecules that can improve the ability of patients' immune cells to attack cancer.

Summary of the Invention

[0008] The present invention provides a method for treating cancer and improving PD-1 / PD-L1-based immunotherapy, including reducing soluble CD28 levels. Agents that bind to membrane CD28 and inhibit the proteolytic cleavage of mCD28, and agents that bind to soluble CD28 and are neither CD28 agonists nor antagonists are also provided, as well as methods for producing these agents.

[0009] 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 soluble CD28 (sCD28) levels in the subject.

[0010] There is provided a method of improving PD-1 and / or PD-L1-based immunotherapy in a subject in need thereof, the method comprising reducing sCD28 levels in the subject.

[0011] According to another aspect, there is provided an agent that binds to soluble CD28 (sCD28) and is neither a CD28 agonist nor an antagonist.

[0012] In some embodiments, the subject in need of immunotherapy has cancer. In some embodiments, the subject does not respond or has a low response to PD-1 and / or PD-L1-based immunotherapy.

[0013] In some embodiments, the reduction occurs in the subject's blood, peripheral blood, or tumor microenvironment. In some embodiments, reducing comprises a. administering to the subject an agent that binds to sCD28, the agent, upon binding, either degrading sCD28 or targeting sCD28 for degradation; b. administering to the subject an inhibitory nucleic acid molecule that binds to the mRNA encoding sCD28 and does not bind to the mRNA encoding membrane CD28 (mCD28); c. administering to the subject an agent that binds to mCD28 and inhibits proteolytic cleavage of mCD28; d. administering to the subject a dimeric peptide comprising the stalk region of human CD28; e. administering to the subject an agent that inhibits a protease capable of cleaving mCD28, and f. comprising at least one of collecting blood from a subject, reducing the amount of sCD28 in the blood, and returning the blood to the subject.

[0014] According to some embodiments, the stalk region a. comprises the amino acid sequence GKHLCPSPLFPGPSKP (SEQ ID NO: 9) or KGKHLCPSPLFPGPS (SEQ ID NO: 36); b. consists of the amino acid sequence HVKGKHLCPSPLFPGPSKP (SEQ ID NO: 10), or c. is both (a) and (b).

[0015] According to some embodiments, the method does not degrade mCD28 or reduce mCD28-mediated immune cell activation.

[0016] According to some embodiments, the subject's blood before the reduction contains at least 5 ng / ml of sCD28.

[0017] 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.

[0018] According to some embodiments, the method of the invention further comprises administering another immunotherapy to the subject. According to some embodiments, the immunotherapy a. is a checkpoint inhibitor; b. is a chimeric antigen receptor (CAR)-based therapy; and c. is selected from cancer vaccines.

[0019] According to some embodiments, the checkpoint inhibitor is an immunotherapy based on PD-1 and / or PD-L1.

[0020] In another aspect, provided is an agent that binds to membrane CD28 (mCD28) and inhibits proteolytic cleavage of mCD28.

[0021] According to some embodiments, the agent is neither a CD28 agonist nor an antagonist. According to some embodiments, the agent neither degrades mCD28 nor inhibits mCD28-mediated immune cell activation.

[0022] According to some embodiments, the agent a. does not induce antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC); b. comprises IgG2 or IgG4; c. comprises an Fc domain designed to reduce CDC, ADCC, or both; d. lacks an Fc domain; e. is a Fab fragment; f. is a single-chain antibody; g. is a single-domain antibody; h. is a small molecule; i. is a peptide that specifically binds to mCD28; or j. is a combination thereof.

[0023] According to some embodiments, the agent binds within the stalk region of CD28. According to some embodiments, the stalk region a. comprises the amino acid sequence GKHLCPSPLFPGPSKP (SEQ ID NO: 9) or KGKHLCPSPLFPGPS (SEQ ID NO: 36); b. consists of the amino acid sequence HVKGKHLCPSPLFPGPSKP (SEQ ID NO: 10); or c. is both (a) and (b).

[0024] According to some embodiments, the agent inhibits proteolytic cleavage by 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 ADAM10 and ADAM17.

[0025] According to some embodiments, the agent is an antibody or an antigen-binding fragment thereof, comprising three heavy-chain CDRs (CDR-H) and three light-chain CDRs (CDR-L), CDR-H1 comprises the amino acid sequence shown in SEQ ID NO: 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).

[0026] According to some embodiments, the agent of the present invention a. a heavy chain comprising the amino acid sequence of SEQ ID NO: 53; and b. at least one light chain comprising the amino acid sequence of SEQ ID NO: 55.

[0027] According to some embodiments, the agent does not bind to membrane CD28 (mCD28).

[0028] According to some embodiments, the agent is selected from an antibody or an antigen-binding fragment thereof, a Fab fragment, a single-chain antibody, a single-domain antibody, a small molecule, and a peptide having specific binding to sCD28.

[0029] According to some embodiments, the binding of the agent to sCD28 in an organism a. results in degradation of the bound sCD28; b. removal of sCD28 from the blood; and c. leads to at least one of transport of the bound sCD28 to lysosomes, endosomes, proteasomes or combinations thereof.

[0030] According to some embodiments, the agent does not inhibit the binding of sCD28 to its ligand. According to some embodiments, the agent binds to dimeric sCD28, monomeric sCD28, or both. According to some embodiments, the agent binds to the outside of the IgV domain of sCD28.

[0031] According to some embodiments, the agent is an antibody or an antigen-binding fragment thereof, and comprises three heavy-chain CDRs (CDR-H) and three light-chain CDRs (CDR-L), CDR-H1 comprises the amino acid sequence shown in SEQ ID NO: 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); 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 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).

[0032] According to some embodiments, the agent of the present invention is a. a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 41, 45 or 49; and b. at least one light chain comprising an amino acid sequence selected from SEQ ID NO: 43, 47 or 51.

[0033] According to some embodiments, the antigen-binding fragment is selected from the group consisting of Fv, Fab, F(ab’)2, scFV or scFV2 fragments.

[0034] According to some embodiments, the agent is humanized.

[0035] According to some embodiments, the agent does not induce antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0036] According to another aspect, a method for producing an agent of the present invention, comprising obtaining an agent that binds to the CD28 extracellular domain 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 culturing a host cell comprising one or more vectors comprising a nucleic acid sequence encoding an agent, wherein the nucleic acid sequence i. obtains an agent that binds to the CD28 extracellular domain or a fragment thereof; ii. tests the ability of the agent to block the cleavage of mCD28 by a protease; and iii. selects at least one agent that blocks the cleavage of mCD28 by a protease selected by, and culturing, thereby providing a method for producing an agent of the present invention.

[0037] According to another aspect, a method for producing an agent of the present invention, comprising Obtaining an agent that binds to the CD28 extracellular domain or a fragment thereof, assaying mCD28 downstream signaling in the presence of the obtained agent, and selecting at least one agent that neither substantially activates nor substantially antagonizes mCD28 signaling; or Culturing a host cell comprising one or more vectors comprising a nucleic acid sequence encoding an agent, wherein the nucleic acid sequence is i. Obtaining an agent that binds to the CD28 extracellular domain or a fragment thereof; ii. Assaying mCD28 downstream signaling in the presence of the obtained agent; and iii. Selecting at least one agent that neither substantially activates nor substantially antagonizes mCD28 signaling A method of culturing is provided, which comprises culturing with an agent selected by the above, thereby producing the agent of the present invention.

[0038] According to some embodiments, the protease is selected from ADAM10 and ADAM17.

[0039] According to some embodiments, obtaining an agent that specifically binds to the CD28 extracellular domain or a fragment thereof is obtaining an agent that specifically binds to the CD28 stalk domain.

[0040] According to some embodiments, the method of the present invention further comprises assaying mCD28 downstream signaling in the presence of the obtained agent and selecting at least one agent that neither substantially activates nor substantially antagonizes mCD28 signaling.

[0041] According to some embodiments, the method is a. Testing the binding of the obtained agent to mCD28 and selecting at least one agent that does not bind to mCD28; and b. further comprising at least one of testing the binding of the obtained agent to sCD28 from cancer patients and selecting at least one agent that binds to sCD28 from cancer patients.

[0042] According to some embodiments, obtaining the agent comprises a. immunizing an organism with the CD28 extracellular domain or a fragment thereof and collecting antibodies from the immunized organism; b. screening a library of agents for binding to the CD28 extracellular domain or a fragment thereof and selecting an agent that binds, comprising at least one of these.

[0043] According to some embodiments, the CD28 extracellular domain or a fragment thereof is a dimer or a monomer.

[0044] According to some embodiments, the organism is selected from rabbits, mice, rats, sharks, camels, chickens, goats, and phages.

[0045] According to some embodiments, collecting the antibodies comprises a. extracting B cells from the spleen of the immunized organism; b. fusing the extracted B cells with myeloma cells to generate hybridomas; and c. collecting antibodies from the hybridomas.

[0046] According to some embodiments, selecting the agent that binds comprises sequencing the selected agent and generating a recombinant form of the agent from the sequence.

[0047] According to another aspect, an agent produced by the method of the present invention is provided.

[0048] According to another aspect, a pharmaceutical composition comprising the agent of the present invention and a pharmaceutically acceptable carrier, excipient, or adjuvant is provided.

[0049] According to another aspect, there is provided a method of treating and / or preventing cancer, or treating and / or preventing cancer in a subject in need of immunotherapy based on PD-1 and / or PD-L1, or improving immunotherapy based on PD-1 and / or PD-L1, the method comprising administering to the subject the pharmaceutical composition of the present invention.

[0050] According to another aspect, there is provided a method of determining the suitability of a subject to be treated by the method of the present invention, the method comprising obtaining 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 suitable for the treatment method of the present invention.

[0051] According to another aspect, there is provided a kit comprising at least one agent of the present invention.

[0052] According to some embodiments, the kit of the present invention a. anti-PD-1 and / or PD-L1 immunotherapy; b. a label indicating that the agent of the present invention is for use in combination with immunotherapy based on PD-1 and / or PD-L1; and c. further comprises at least one secondary detection molecule for detecting at least one agent according to any one of claims 15 to 37 and 49.

[0053] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description, and thus, the detailed description and the specific examples are to be construed as illustrative only and not as limiting, while showing preferred embodiments of the present invention.

Brief Description of the Drawings

[0054]

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BRIEF DESCRIPTION OF THE INVENTION

[0055] In some embodiments, the present invention provides an agent that binds to membrane CD28 (mCD28) and inhibits proteolytic cleavage of mCD28, and an agent that binds to soluble CD28 (sCD28) and is neither a CD28 agonist nor an antagonist. The present invention further provides an agent that, upon binding, degrades sCD28, or results in the degradation of sCD28, or results in its clearance from the circulation, tissues, and / or tumor microenvironment (TME). In some cases, the agent can perform multiple of these tasks. Also provided is a method of treating cancer and improving PD-1 / PD-L1-based immunotherapy, including reducing soluble CD28 (sCD28) levels. The agents and methods of the present invention are based on the surprising finding that a large number of cancer patients have elevated sCD28 levels in their bloodstream. sCD28 is a known immunomodulator that is sometimes overexpressed in autoimmune diseases. However, very high levels have never been reported in a wide variety of cancers until now. Furthermore, it has unexpectedly been discovered that sCD28 can inhibit PD-1 / PD-L1-based immunotherapy. Thus, reduction of sCD28 in the bloodstream of a subject leads to an increase in the subject's ability to fight cancer and a reduction in the detrimental effects of sCD28 on the effectiveness of immunotherapy.

[0056] The anti-cleavage molecule of the present invention has a dual advantage. By blocking proteolytic cleavage of mCD28, the amount of CD28 on the cell surface of T cells is maintained high. This enables rapid and effective activation of T cells, which is impaired when the level of surface CD28 is reduced by cleavage. Furthermore, reduction of cleavage leads to a reduction of sCD28 in the bloodstream of the subject, and thus to a reduction of the detrimental effects of sCD28 on the subject's ability to fight cancer and the effectiveness of immunotherapy.

[0057] Binding of sCD28 According to a first aspect, there is provided an agent that binds to soluble CD28 (sCD28) and is neither a CD28 agonist nor an antagonist.

[0058] In some embodiments, CD28 is mammalian CD28. In some embodiments, CD28 is human CD28. In some embodiments, human CD28 has the amino acid sequence: MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 1), or consists of the same. In some embodiments, mature CD28 lacks a signal peptide and has the sequence: NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 2).

[0059] In some embodiments, the DNA coding sequence encoding full-length human CD28 comprises the sequence: ATGCTCAGGCTGCTCTTGGCTCTCAACTTATTCCCTTCAATTCAAGTAACAGGAAACAAGATTTTGGTGAAGCAGTCGCCCATGCTTGTAGCGTACGACAATGCGGTCAACCTTAGCTGCAAGTATTCCTACAATCTCTTCTCAAGGGAGTTCCGGGCATCCCTTCACAAAGGACTGGATAGTGCTGTGGAAGTCTGTGTTGTATATGGGAATTACTCCCAGCAGCTTCAGGTTTACTCAAAAACGGGGTTCAACTGTGATGGGAAATTGGGCAATGAATCAGTGACATTCTACCTCCAGAATTTGTATGTTAACCAAACAGATATTTACTTCTGCAAAATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCTGA (SEQ ID NO: 3).

[0060] As used herein, sCD28 refers to any CD28 fragment or variant that does not contain a transmembrane domain and thus cannot be incorporated into the 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 the blood. In some aspects, sCD28 is CD28 in the TME. In some aspects, sCD28 is CD28 in the body fluid. In some embodiments, sCD28 lacks exon 3 of CD28. In some embodiments, sCD28 is a splice variant resulting from alternative splicing that splices exon 3 of CD28. In some embodiments, sCD28 is a cleavage product from membrane CD28 (mCD28). In some embodiments, sCD28 is a truncated CD28. In some embodiments, sCD28 lacks the cytoplasmic domain of full-length CD28. In some embodiments, sCD28 is dimeric sCD28. In some embodiments, sCD28 is monomeric sCD28. In some embodiments, sCD28 is not a splice variant resulting from alternative splicing of CD28. In some aspects, alternative splicing splices exon 3 of CD28. In some embodiments, sCD28 comprises the amino acid sequence: MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGEE (SEQ ID NO: 5).In some embodiments, sCD28 lacks a signal peptide and comprises the sequence: NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGEE (SEQ ID NO: 6).

[0061] In some embodiments, the DNA coding sequence encoding human sCD28 comprises the sequence: ATGCTCAGGCTGCTCTTGGCTCTCAACTTATTCCCTTCAATTCAAGTAACAGGAAACAAGATTTTGGTGAAGCAGTCGCCCATGCTTGTAGCGTACGACAATGCGGTCAACCTTAGCTGCAAGTATTCCTACAATCTCTTCTCAAGGGAGTTCCGGGCATCCCTTCACAAAGGACTGGATAGTGCTGTGGAAGTCTGTGTTGTATATGGGAATTACTCCCAGCAGCTTCAGGTTTACTCAAAAACGGGGTTCAACTGTGATGGGAAATTGGGCAATGAATCAGTGACATTCTACCTCCAGAATTTGTATGTTAACCAAACAGATATTTACTTCTGCAAAATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCTGA (SEQ ID NO: 7).

[0062] In some embodiments, the agent binds to sCD28 and mCD28 and leads to or causes the degradation or clearance of sCD28 only. In some embodiments, the agent does not bind to membrane CD28 (mCD28). As used herein, mCD28 refers to any CD28 that contains a transmembrane domain and can thus be incorporated 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 within the membrane. In some embodiments, mCD28 passes through the ER, passes through the Golgi apparatus, and reaches the plasma membrane of the cell. In some embodiments, mCD28 is within the plasma membrane of an immune cell. In some embodiments, mCD28 is within the plasma membrane of a T cell.

[0063] 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 binder is also an mCD28 agonist.

[0064] The term "agonist" generally refers to a molecule, compound, or agent that binds to a receptor and fully or partially activates the receptor. In some embodiments, the agonist binds at the same site as the natural ligand. In some embodiments, the agonist binds at an allosteric site different from the binding site of the natural ligand. The term "antagonist" generally refers to a molecule, compound, or agent that binds to a receptor at the same site or a different site than the agonist, does not activate the receptor, and performs one or more of interfering with or blocking the activation of the receptor by the natural ligand and interfering with or blocking the activation of the receptor by a receptor agonist. In some embodiments, the antibodies of the present invention bind to mCD28 but do not activate the receptor or block the activation of the receptor. In some embodiments, they do not block activation by CD86. In some embodiments, the antibodies of the present invention do not bind to mCD28.

[0065] As used herein, "direct agonist / antagonist" refers to a molecule that binds to a receptor (mCD28) and, by binding, increases / decreases signal transduction by that molecule. In the case of mCD28, an agonist binds to mCD28 and, by binding, increases intracellular mCD28 signal transduction. In some embodiments, the agonist increases T cell activation. In some embodiments, the agonist increases T cell proliferation. In some embodiments, the agonist increases pro-inflammatory cytokine secretion. Pro-inflammatory cytokines are well known in the art and are known to be secreted by activated T cells. Examples of pro-inflammatory cytokines include, but are not limited to, TNFα, IFNγ, IL-1B, and IL-6. In some embodiments, the pro-inflammatory cytokine is IFNγ. In the case of mCD28, an antagonist binds to mCD28 and, by binding, decreases mCD28 signal transduction in the cell. In some embodiments, the antagonist decreases T cell activation, decreases T cell proliferation, and / or decreases pro-inflammatory cytokine secretion. Molecules that affect receptor signal transduction by contacting a ligand, contacting an inhibitor, contacting a co-receptor, or contacting a molecule other than the receptor in question to modify receptor signal transduction are not considered direct agonists / antagonists. In some embodiments, the agents of the invention contact sCD28 in serum, thereby enabling an increase in signal transduction via mCD28 on the cell. As a result, mCD28 signal transduction increases, but since binding to mCD28 does not increase receptor signal transduction, the antibody is not an mCD28 agonist or a direct agonist.

[0066] In some embodiments, the agent does not bind to the ligand-binding domain of mCD28. In some embodiments, the agent does not occlude or block access to the ligand-binding domain. In some embodiments, the agent does not bind to the IgV domain of sCD28, does not occlude or block access to the domain. In some embodiments, the IgV domain is the ligand-binding domain. In some embodiments, the ligand-binding domain comprises amino acids 28-137 of SEQ ID NO: 1. In some embodiments, the ligand-binding domain comprises or consists of the amino acid sequence MLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKG (SEQ ID NO: 8). In some embodiments, the agent does not inhibit the binding of sCD28 to its ligand. In some embodiments, the CD28 ligand is selected from CD80, CD86 and ICOS-L. In some embodiments, the CD28 ligand is CD86. In some embodiments, the CD28 ligand is CD80. In some embodiments, the CD28 ligand is ICOS-L. In some embodiments, CD86 is CD86-Fc. In some embodiments, CD80 is CD80-Fc.

[0067] In some embodiments, the agent binds to the stalk region of CD28. In some embodiments, the agent binds to the membrane-proximal region of mCD28. In some embodiments, the stalk region comprises the sequence GKHLCPSPLFPGPSKP (SEQ ID NO:9). In some embodiments, the stalk region comprises the sequence KGKHLCPSPLFPGPS (SEQ ID NO:36). In some embodiments, the stalk region comprises, or consists of, the sequence HVKGKHLCPSPLFPGPSKP (SEQ ID NO:10). In some embodiments, the agent binds to monomeric sCD28. In some embodiments, the agent binds to dimeric sCD28. In some embodiments, the agent binds to monomeric sCD28, dimeric sCD28, or both. In some embodiments, the agent binds to monomeric CD28 but not to dimeric CD28. Since functional mCD28 is a dimer, binding to only CD28 monomers can be used to ensure that the agent does not bind to mCD28.

[0068] Examples of agents include, but are not limited to, antibodies, antigen-binding fragments of antibodies, nanobodies, single-chain antibodies, single-domain antibodies, small molecules, peptides, and DARPins. In some embodiments, the agent is selected from an antibody, an antigen-binding fragment of an antibody, a Fab fragment, a nanobody, a single-chain antibody, a single-domain antibody, a small molecule, a peptide, and a DARPin. In some embodiments, the agent is selected from an antibody, an antigen-binding fragment of an antibody, a Fab fragment, a single-chain antibody, a single-domain antibody, a small molecule, and a peptide that specifically binds to CD28. In some embodiments, the agent is a single-domain antibody. In some embodiments, the agent is a nanobody. In some embodiments, the agent is a VHH antibody. As used herein, the terms "single-domain antibody," "nanobody," and "VHH antibody" are synonymous and used interchangeably. In some embodiments, the peptide has specific binding to CD28. In some embodiments, the agent is a peptide having specific binding to CD28. In some embodiments, the peptide is selected from an antibody, an antigen-binding fragment of an antibody, a Fab fragment, a single-chain antibody, a single-domain antibody, a nanobody, a VHH antibody, and an antibody mimetic. As used herein, the term "antibody mimetic" refers to an organic compound capable of specifically binding to a target antigen. In some embodiments, the antibody mimetic is not structurally related to an antibody. Examples of antibody mimetics include, but are not limited to, affilins, affimers, affitins, alphabodies, anticalins, avimers, DARPins, finomers, Kunitz domain peptides, monobodies, and nanoclamps. In some embodiments, the antibody mimetic is a DARPin. All of these agents are well known in the art and are known to be useful for blocking the interaction between receptors and their ligands. Small molecules and proteins that can bind to mCD28 can shield the cleavage site, or cause protease impairment, or impair its access. In some embodiments, the protein is an antibody mimetic. As used herein, the term "DARPin" refers to a designed ankyrin repeat protein.DARPin is a genetically engineered antibody mimetic protein and is generally highly specific for protein targets. Thus, a DARPin of CD28 is an example of an agent.

[0069] In some embodiments, the agent that binds to sCD28 is an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody to sCD28 is a single-domain antibody. In some embodiments, the antibody to sCD28 lacks an Fc domain. In some embodiments, the agent that binds to sCD28 is an antigen-binding domain lacking an Fc domain. In some embodiments, the agent that binds to sCD28 is a single-domain antibody. In some embodiments, the agent that binds to sCD28 is a camelid, shark, or nanobody. In some embodiments, the antibody or fragment is fused to another protein or a fragment of a protein. In some embodiments, the second protein or fragment particularly increases the half-life in serum. In some embodiments, the half-life extending protein is human serum albumin. In some embodiments, the agent is modified by a chemical that produces a modification that improves the half-life. In some embodiments, the modification is pegylation and the chemical is polyethylene glycol. It is understood by those skilled in the art that any half-life extending protein or chemical, or modification known in the art can be used.

[0070] As used herein, the term "antibody" refers to a polypeptide or group of polypeptides comprising at least one binding domain formed from the folding of a polypeptide chain having a three-dimensional binding space with a charge distribution complementary to the internal surface shape and the characteristics of the antigenic determinant of an antigen. Antibodies typically have a tetrameric form comprising two identical pairs of polypeptide chains, each pair having one "light" chain and one "heavy" chain. The variable regions of each light / heavy chain pair form the antibody binding site. Antibodies include oligoclonal, polyclonal, monoclonal, chimeric, camelized, CDR-grafted, multispecific, bispecific, catalytic, humanized, fully human, anti-idiotype, and soluble or bound and labelable antibodies, alone or in combination with other amino acid sequences, as well as epitope-binding fragments, variants or derivatives thereof. Antibodies may be from any species. The term antibody also includes binding fragments including, but not limited to, Fv, Fab, Fab’, F(ab’)2 single-chain antibody (scFv), dimeric variable region (diabody), and disulfide-bonded variable region (dsFv). In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing an antigen-binding site. Antibody fragments may or may not be fused to another immunoglobulin domain including, but not limited to, the Fc region or fragments thereof. It is further understood by those skilled in the art that other fusion products can be generated including, but not limited to, scFv-Fc fusions, variable region (e.g., VL and VH)-Fc fusions and scFv-scFv-Fc fusions.

[0071] Immunoglobulin molecules may be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. In some embodiments, the antibody comprises IgG2 or IgG4. In some aspects, the antibody comprises IgG2. In some aspects, the antibody comprises IgG4.

[0072] The basic unit of a naturally occurring antibody structure is a heterotetrameric glycoprotein complex of approximately 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains, which are joined by both non-covalent and disulfide bonds. Each heavy and light chain also has regularly spaced intra-chain disulfide cross-links. There are five human antibody classes (IgG, IgA, IgM, IgD, IgE), and the various subclasses within these classes are recognized based on differences in structure such as the number of immunoglobulin units within a single antibody molecule, the disulfide cross-link structure of the individual units, as well as differences in chain length and sequence. The class and subclass of an antibody is its isotype.

[0073] The amino-terminal regions of the heavy and light chains are more diverse in sequence than the carboxy-terminal regions and are thus called variable domains. This part of the antibody structure confers the antigen-binding specificity of the antibody. The heavy variable (VH) domain and the light variable (VL) domain together form a single antigen-binding site, and thus there are two antigen-binding sites in a basic immunoglobulin unit. Certain amino acid residues are thought to form the interface between the light-chain variable domain and the heavy-chain variable domain (Chothia et al., J. Mol. Biol. 186, 651-63 (1985); Novotny and Haber, (1985) Proc. Natl. Acad. Sci. USA 82 4592-4596).

[0074] The carboxy-terminal portions of the heavy and light chains form the constant domains, namely CH1, CH2, CH3, CL. The diversity of these domains is much lower, but there are differences among animal species, and there are also several different antibody isotypes that have different functions within the same individual.

[0075] The term "framework region" or "FR" refers to amino acid residues within the variable domain of an antibody other than the hypervariable region amino acid residues defined herein. The term "hypervariable region" when used herein refers to amino acid residues within the variable domain of an antibody that are involved in antigen binding. The hypervariable region includes amino acid residues from "complementary determining regions" or "CDRs". CDRs are primarily involved in binding to the epitope of an antigen. The ranges of FRs and CDRs are precisely defined (see Kabat et al.).

[0076] Immunoglobulin variable domains can also be analyzed using the IMGT information system (www: / / imgt.cines.fr / ) (IMGT® / V-Quest) to identify variable region segments that include CDRs. See, for example, Brochet, X. et al., Nucl. Acids Res. 36:W503-508 (2008).

[0077] Chothia et al. also defined a numbering system for variable domain sequences applicable to any antibody. One of ordinary skill in the art can clearly assign this "Chothia numbering" system to any variable domain sequence without relying on experimental data beyond the sequence itself. As used herein, "Chothia numbering" refers to the numbering system described in Chothia et al., Journal of Molecular Biology, "Canonical Structures for the Hypervariable regions of immunoglobulins" (1987) and Chothia et al., Nature, "Conformations of Immunoglobulin Hypervariable Regions" (1989).

[0078] As used herein, the term "humanized antibody" refers to an antibody derived from a non-human species whose protein sequence has been modified to enhance its similarity to a human antibody. A humanized antibody can be produced by generating recombinant DNA encoding the CDRs of a non-human antibody surrounded by sequences similar to those of a human antibody. In some embodiments, a humanized antibody is a chimeric antibody. In some embodiments, humanization involves inserting the CDRs of the invention into a human antibody scaffold or framework. Humanized antibodies are well known in the art, and any method for generating them that retains the CDRs of the invention can be used.

[0079] The term "monoclonal antibody" or "mAb" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible variants that may arise during the production of the monoclonal antibody, and / or bind to the same epitope, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of the antibody being obtained from a substantially homogeneous population of antibodies and should not be construed as being produced by any particular method of preparation. Monoclonal antibodies used in accordance with the methods provided herein may be made by the hybridoma method first described by Kohler et al, Nature 256:495 (1975), or 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).

[0080] The mAbs of the present invention may be of any immunoglobulin class, including IgG, IgM, IgD, IgE, or IgA. The hybridomas that produce the mAbs can be cultured in vitro or in vivo. High-titer mAbs can be obtained by in vivo production, where cells from individual hybridomas are intraperitoneally injected into pristane-primed Balb / c mice, generating ascites containing high concentrations of the desired mAbs. mAbs of isotype IgM or IgG can be purified from such ascites or from culture supernatants using column chromatography methods well known to those skilled in the art.

[0081] An "antibody fragment" includes a portion of an intact antibody and preferably includes its antigen-binding region. Examples of antibody fragments include Fab, Fab’, F(ab’)2, and Fv fragments; diabodies; tandem diabodies (taDb), linear antibodies (e.g., U.S. Patent No. 5,641,870, Example 2; Zapata et al, Protein Eng. 8(10):1057-1062 (1995)); single-arm antibodies, single variable domain antibodies, minibodies, single-chain antibody molecules; multispecific antibodies formed from antibody fragments (e.g., Db-Fc, taDb-Fc, taDb-CH3, (scFV)4-Fc, di-scFv, bi-scFv, or tandem (di, tri)-scFv); and Bispecific T cell engagers (BiTE).

[0082] Papain digestion of an antibody produces two identical antigen-binding fragments called "Fab" fragments, each having a single antigen-binding site and a residual "Fc" fragment, the name of which reflects its ability to readily crystallize. Pepsin treatment produces an F(ab’)2 fragment that has two antigen-binding sites but can cross-link antigens.

[0083] "Fv" is the smallest antibody fragment that contains the complete antigen recognition and antigen-binding site. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain that are firmly covalently bound. Three surfaces of the VH-VL dimer are in this configuration. Collectively, six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of the Fv containing only three hypervariable regions specific for the antigen), although having a lower affinity than the entire binding site, has the ability to recognize and bind the antigen.

[0084] 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 several residues are added to the carboxy terminus of the heavy-chain CH1 domain that contains one or more cysteines from the antibody hinge region. Fab’-SH is, in the present specification, the designation for Fab’ in which the cysteine residue(s) of the constant domain retain at least one free thiol group. The F(ab’)2 antibody fragment was originally generated as a pair of Fab’ fragments with a hinge cysteine in between. Other chemical couplings of antibody fragments are also known.

[0085] The "light chain" of an antibody (immunoglobulin) from a vertebrate species can be assigned to one of two clearly distinguishable types called κ and λ based on the amino acid sequence of the constant domain.

[0086] Antibodies can be assigned to different classes according to the amino acid sequence of their heavy-chain constant domains. Intact antibodies have five major classes: IgA, IgD, IgE, IgG, and IgM, and some of these can be further classified into subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains corresponding to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional arrangements of the different classes of immunoglobulins are well known.

[0087] A "single-chain Fv" or "scFv" antibody fragment contains the VH and VL domains of an antibody, and these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH domain and the VL domain that enables the scFv to form the desired structure for antigen binding. For a discussion of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenberg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0088] The term "diabody" refers to a small antibody fragment having two antigen-binding sites that contains a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) of the same polypeptide chain (VH-VL). By using a linker that is too short to permit pairing between the two domains of the same chain, the domains pair with the complementary domains of the other chain to form two antigen-binding sites. The generation of diabodies is known in the art and is described in Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0089] The term "multispecific antibody" is used in the broadest sense and encompasses, in particular, antibodies having polyepitope specificity. Such multispecific antibodies include antibodies in which the VHVL units contain heavy-chain variable domains (VH) and light-chain variable domains (VL) having polyepitope specificity, antibodies having two or more VL and VH domains in which each VHVL unit binds to a different epitope, antibodies having two or more single variable domains in which each single variable domain binds to a different epitope, full-length antibodies, Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies, triabodies, trifunctional antibodies, antibody fragments such as covalently or non-covalently linked antibody fragments, etc., but are not limited thereto. "Polyepitope specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different target(s).

[0090] The monoclonal antibodies of the present invention can be prepared using methods well 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).

[0091] In addition to conventional methods of producing antibodies in vivo, antibodies can be generated in vitro using phage display technology. The generation of such recombinant antibodies is much faster compared to conventional antibody generation, and they can be generated against a vast number of antigens. Furthermore, when using conventional methods, many antigens have been found to be non-immunogenic or highly toxic and thus cannot be used for antibody generation in animals. Additionally, affinity maturation of recombinant antibodies (i.e., increase in affinity and specificity) is very straightforward and relatively fast. Finally, a large number of different antibodies against a particular antigen can be generated in a single selection procedure. To generate recombinant monoclonal antibodies, large pools of antibodies with different antigen recognition sites can be generated using various methods based on display libraries. Such libraries can be created in several ways: by cloning synthetic CDR3 regions into a pool of heavy-chain germline genes to generate a synthetic repertoire, a large antibody repertoire can be generated from which recombinant antibody fragments with various specificities can be selected. Human lymphocyte pools can be used as starting materials for antibody library construction. It is possible to construct a naive repertoire of human IgM antibodies and thus create a highly diverse human library. This method has been widely and successfully used to select a large number of antibodies against various antigens. Protocols for bacteriophage library construction and selection of recombinant antibodies are described in the well-known reference text Current Protocols in Immunology, Colligan et al (Eds.), John Wiley & Sons, Inc. (1992 - 2000), Chapter 17, Section 17.1.

[0092] Non-human antibodies can be humanized by any method known in the art. In one method, non-human complementarity-determining regions (CDRs) are inserted into a human antibody or consensus antibody framework sequence. Further modifications can then be introduced into the antibody framework to modulate affinity or immunogenicity.

[0093] In some embodiments, antibodies and portions thereof include antibodies, antibody fragments, Fab and F(ab’)2, single domain antigen-binding recombinant fragments, and natural nanobodies. In some embodiments, the antigen-binding fragment is an Fv, Fab, F(ab’) 2 , scFV or scFV 2 fragment selected from the group consisting of

[0094] In some embodiments, the invention provides a nucleic acid sequence encoding an antibody or antigen-binding portion of the invention.

[0095] For example, the polynucleotide can encode an entire immunoglobulin molecule chain, such as a light or heavy chain. A complete heavy chain includes not only the heavy chain variable region (VH), but also the heavy chain constant region (CH) (typically including three constant regions, CH1, CH2, and CH3); and a “hinge” region. In some situations, the presence of the constant region is desirable.

[0096] Other polypeptides that can be encoded by the polynucleotide include antigen-binding antibody fragments such as single domain antibodies ("dAbs"), Fv, scFv, Fab', and CHI, and the CK or CL domain has been excised. Since minibodies are smaller than conventional antibodies, they should have improved tissue penetration for use in clinical / diagnostic applications, but being bivalent, they should retain a higher binding affinity than monovalent antibody fragments such as dAbs. Thus, unless the context indicates otherwise, the term "antibody" as used herein encompasses not only the whole antibody molecule but also antigen-binding antibody fragments of the types discussed above. Each framework region present in the encoded polypeptide can include at least one amino acid substitution compared to the corresponding human acceptor framework. Thus, for example, the framework region can include a total of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions relative to the acceptor framework region. Considering the properties of the individual amino acids that make up the disclosed protein products, some reasonable substitutions will be recognized by those skilled in the art. Amino acid substitutions, i.e., "conservative substitutions," can be made, for example, based on the similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues involved.

[0097] Suitably, the polynucleotides described herein can be isolated and / or purified. In some embodiments, the polynucleotide is an isolated polynucleotide.

[0098] As used herein, the term "not naturally occurring" substances, compositions, entities, and / or any combination of substances, compositions or entities, or grammatical variations thereof, explicitly excludes those forms of substances, compositions, entities, and / or any combination of substances, compositions or entities that are well understood by those skilled in the art to be "naturally occurring," or that are determined or interpreted, or can be determined or interpreted, to be "naturally occurring" by a trier or administrative or judicial body at any point in time, but is a conditional term for exclusion only.

[0099] 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).

[0100] 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).

[0101] In some embodiments, the agent is an antibody or an antigen-binding portion thereof that includes three heavy-chain CDRs (CDR-H) and three light-chain CDRs (CDR-L), wherein CDR-H1 includes the amino acid sequence set forth in SEQ ID NO: 24 (GYTFTDY), CDR-H2 includes the amino acid sequence set forth in SEQ ID NO: 25 (NPNYDS), CDR-H3 includes the amino acid sequence set forth in SEQ ID NO: 26 (SSPYYDSNHFDY), CDR-L1 includes the amino acid sequence set forth in SEQ ID NO: 27 (SARSSINYMH), CDR-L2 includes the amino acid sequence set forth in SEQ ID NO: 28 (DTSKLAS), and CDR-L3 includes the amino acid sequence set forth in SEQ ID NO: 29 (HQRNSYPFT).

[0102] In another aspect, an antibody or an antigen-binding portion thereof that includes three heavy-chain CDRs (CDR-H) and three light-chain CDRs (CDR-L) is provided, wherein CDR-H1 includes the amino acid sequence set forth in SEQ ID NO: 12 (GYTLTNY), CDR-H2 includes the amino acid sequence set forth in SEQ ID NO: 13 (NTYTGK), CDR-H3 includes the amino acid sequence set forth in SEQ ID NO: 14 (GDANQQFAY), CDR-L1 includes the amino acid sequence set forth in SEQ ID NO: 15 (KASQDINSYLS), CDR-L2 includes the amino acid sequence set forth in SEQ ID NO: 16 (RANRLVD), and CDR-L3 includes the amino acid sequence set forth in SEQ ID NO: 17 (LQYDEFPPT).

[0103] In another aspect, an antibody or an antigen-binding portion thereof that includes three heavy-chain CDRs (CDR-H) and three light-chain CDRs (CDR-L) is provided, wherein CDR-H1 includes the amino acid sequence set forth in SEQ ID NO: 18 (GYTFTSY), CDR-H2 includes the amino acid sequence set forth in SEQ ID NO: 19 (YPGDGD), CDR-H3 includes the amino acid sequence set forth in SEQ ID NO: 20 (NYRYSSFGY), CDR-L1 includes the amino acid sequence set forth in SEQ ID NO: 21 (KSSQSLLNSGNQKNYLT), CDR-L2 includes the amino acid sequence set forth in SEQ ID NO: 22 (WASTRES), and CDR-L3 includes the amino acid sequence set forth in SEQ ID NO: 23 (QSDYSYPLT).

[0104] In another aspect, an antibody or antigen-binding portion thereof comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L) is provided, 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).

[0105] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence QIQLVQSGPELKKPGETVKISCKASGYTLTNYGMNWVKQAPGKGLKWMGWINTYTGKPTYVDDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARGDANQQFAYWGQGTLVTVS (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 aspects, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence QIQLVQSGPELKKPGETVKISCKASGYTLTNYGMNWVKQAPGKGLKWMGWINTYTGKPTYVDDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARGDANQQFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 42). In some embodiments, the heavy chain consists of SEQ ID NO: 42. Antibody #1 referred to in this 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 scheme are SEQ ID NOs: 12-14.

[0106] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the 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 amino acid sequence DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYDDMGIYYCLQYDEFPPTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 44). In some embodiments, the light chain consists of SEQ ID NO: 44. Antibody #1 referred to in this application 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 scheme are SEQ ID NOs: 15-17.

[0107] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence QVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWIKKRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSTTAYMQLSSLASEDSAVYFCARNYRYSSFGYWGQGTLVTVSA (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 aspects, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence QVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWIKKRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSTTAYMQLSSLASEDSAVYFCARNYRYSSFGYWGQGTLVTVSAAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK (SEQ ID NO: 46). In some embodiments, the heavy chain consists of SEQ ID NO: 46. Antibody #2 referred to in this 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 scheme are SEQ ID NOs: 18-20.

[0108] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence DIVMTQSPSSLTVTAGEKVTLSCKSSQSLLNSGNQKNYLTWYQQKPGQPPQLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQSDYSYPLTFGAGTKLELK (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 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 referred to in this 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 scheme are SEQ ID NOs: 21-23.

[0109] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence EVQLQQFGAELVKPGASVKISCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPNYDSTAYNQKFMGKATLTVDKSSNTAYMELRSLTSEDTAVYYCARSSPYYDSNHFDYWGQGTSLTVSS (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 aspects, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence EVQLQQFGAELVKPGASVKISCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPNYDSTAYNQKFMGKATLTVDKSSNTAYMELRSLTSEDTAVYYCARSSPYYDSNHFDYWGQGTSLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 50). In some embodiments, the heavy chain consists of SEQ ID NO: 50. Antibody #3 referred to in this application was sequenced and found to have a heavy chain consisting of SEQ ID NO: 50. The CDRs of this heavy chain determined using the Chothia scheme are SEQ ID NOs: 24-26.

[0110] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the 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 aspects, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence QIVLTQSPAIMSASPGEKVTMTCSARSSINYMHWFQQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISNMEAEDAATYYCHQRNSYPFTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 52). In some embodiments, the light chain consists of SEQ ID NO: 52. Antibody #3 referred to in this 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 scheme are SEQ ID NOs: 27-29.

[0111] 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 a dimer. In some embodiments, the agent binds as a dimer but does not bind to 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 binds to only 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.

[0112] In some embodiments, the antibody or antigen-binding fragment thereof 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 aspects, 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 antigen-binding fragment thereof is an anti-soluble CD28 (sCD28) antibody. An "anti-CD28 antibody", "antibody that recognizes CD28", or "antibody against CD28" is an antibody that binds to 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.

[0113] As used herein, the terms "increased binding affinity" and "greater binding affinity" are interchangeable. In some embodiments, the antibody or antigen-binding portion thereof of the invention has a greater binding affinity for sCD28 compared to mCD28. In one embodiment, the greater affinity as used herein is 10%. In one embodiment, the greater affinity as used herein is 30%. In one embodiment, the greater affinity as used herein is 50%. In one embodiment, the greater affinity as used herein is 75%. In one embodiment, the greater affinity as used herein is 100%. In one embodiment, the greater affinity as used herein is 150%. In one embodiment, the greater affinity as used herein is 250%. In one embodiment, the greater affinity as used herein is 500%. In one embodiment, the greater affinity as used herein is 1,000%. In one embodiment, the greater affinity as used herein is 1.5-fold. In one embodiment, the greater affinity as used herein is 2-fold. In one embodiment, the greater affinity as used herein is 5-fold. In one embodiment, the greater affinity as used herein is 10-fold. In one embodiment, the greater affinity as used herein is 50-fold. In one embodiment, the greater affinity as used herein is 100-fold. In one embodiment, the greater affinity as used herein is 500-fold. In one embodiment, the greater affinity as used herein is 1,000-fold.

[0114] An "antigen" is a molecule or portion of a molecule that elicits antibody formation and can be bound by an antibody. An antigen can have one or more epitopes. The specific reaction described above indicates that the antigen reacts with its corresponding antibody in a highly selective manner and does not react with many other antibodies that may be induced by other antigens.

[0115] As used herein, the terms "antigenic determinant" or "epitope" refer to the region of an antigen molecule that specifically reacts with a particular antibody. Peptide sequences derived from an epitope can be used alone or in combination with a carrier moiety to immunize an animal and generate additional polyclonal or monoclonal antibodies by applying methods known in the art.

[0116] In some embodiments, the antibody comprises N-linked glycosylation at N304 of the heavy chain.

[0117] 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, shark, or nanobody. In some embodiments, the antibody or fragment is fused to another protein or protein fragment. In some embodiments, the second protein or fragment particularly increases the half-life in serum. In some embodiments, the half-life extending protein is human serum albumin. In some embodiments, the agent is modified by a chemical that produces a modification that improves the half-life. In some embodiments, the modification is pegylation and the chemical is polyethylene glycol. It will be understood by those skilled in the art that any half-life extending protein or chemical, or modification known in the art can be used.

[0118] In some embodiments, the binding of the agent to sCD28 degrades sCD28. In some embodiments, the binding of the agent to sCD28 leads to or results in the degradation of sCD28. In some embodiments, the degradation occurs when the binding is within an organism. In some embodiments, the degradation occurs when the binding is in the bloodstream of an organism. In some embodiments, the degradation occurs when the binding is in the TME of an organism. In some embodiments, the degradation includes the removal of sCD28 from the blood. In some embodiments, the degradation includes the transport of the bound sCD28 to lysosomes, endosomes, proteasomes, or combinations thereof. Each possibility represents a separate embodiment of the invention.

[0119] In some embodiments, the binding of the agent to sCD28 in an organism results in the removal of sCD28 from the blood. In some embodiments, the binding of the agent to sCD28 in an organism results in the clearance of sCD28 from the blood. In some embodiments, the binding of the agent to sCD28 in an organism results in the removal of sCD28 from the TME. In some embodiments, the binding of the agent to sCD28 in an organism results in the clearance of sCD28 from the TME. In some embodiments, the binding of the agent to sCD28 in an organism results in the removal of sCD28 from the blood, the TME, or both. In some embodiments, the binding of the agent to sCD28 in an organism results in the clearance of sCD28 from the blood, the TME, or both. In some embodiments, sCD28 is not degraded but is removed from the blood, the TME, or both. In some embodiments, the bound sCD28 is an immune complex.

[0120] There are many known mechanisms for removing bound antigen from the bloodstream. These include, but are not limited to, complement-mediated clearance, phagocytosis by monocytes and macrophages, opsonization, proteolysis, passive diffusion, and active transport. In some embodiments, the active transport is by erythrocytes. In some embodiments, the bound antigen is transported to phagocytic cells. In some embodiments, the bound antigen is transported to lysosomes. In some embodiments, the bound antigen is transported to lysosomes, endosomes, proteasomes, or combinations thereof. Any agent that can induce removal of the bound sCD28 complex can be used in the present invention.

[0121] In some embodiments, the agent reduces the sCD28 level 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 invention. In some embodiments, the agent reduces the sCD28 level to the level of a healthy individual. In some embodiments, the agent reduces the sCD28 level to a maximum of 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 invention. In some embodiments, the agent reduces the blood concentration of sCD28 to a maximum of 5 ng / ml. In some embodiments, the agent reduces the blood concentration of sCD28 to a maximum of 10 ng / ml. In some embodiments, the agent reduces the blood concentration of sCD28 to a maximum of 20 ng / ml. In some embodiments, the agent reduces the sCD28 level to the level of a healthy individual. In some embodiments, the agent reduces the sCD28 level 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 invention. In some embodiments, the agent reduces the sCD28 level to less than 5 ng / ml. In some embodiments, the agent reduces the sCD28 level to less than 10 ng / ml. In some embodiments, the agent reduces the sCD28 level to less than 20 ng / ml. In some embodiments, the reduction or decrease occurs in the blood, peripheral blood or TME of the subject. In some embodiments, the reduction or decrease occurs in the blood.

[0122] In some embodiments, the sCD28 level is as 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 Bender MedSystems ELISA kit BMS290. In some embodiments, the ELISA is performed using an agent of the invention.

[0123] In some embodiments, the agent is a scavenging antibody. As used herein, the term "scavenging antibody" refers to any antibody or antigen-binding fragment thereof that reduces the amount of a soluble component from a solution. In some embodiments, the scavenging antibody does not induce antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the scavenging antibody does not induce complement-dependent cytotoxicity (CDC). In some embodiments, the scavenging antibody does not induce ADCC and / or CDC. In some embodiments, the scavenging antibody comprises an IgG2 or IgG4 domain. In some embodiments, the scavenging antibody comprises an IgG2 domain. In some embodiments, the scavenging antibody comprises an IgG4 domain. In some embodiments, the scavenging antibody comprises an IgG1 or IgG3 that has been mutated to reduce cell death mediated by antibody binding. In some embodiments, the mutation mutates the Fc receptor binding domain. In some embodiments, the Fc domain of the antibody is engineered or mutated to reduce CDC, ADCC, or both. Fc engineering is well known in the art and any mutation or amino acid change known to reduce antibody-mediated cell killing can be used.

[0124] 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 IgG1 or IgG3 that contains a mutation that reduces ADCC, CDC, or both induced by the binding of the antibody. In some embodiments, the mutation reduces ADCC, CDC, or both to zero. ADCC and CDC are well characterized, and antibody sequences that enable the induction of these cytotoxic pathways are well known. Mutations such as non-limiting examples are well known mutations of IgG1 or IgG3 to IgG2 or IgG4. Any such mutations can be used in the framework of the antibodies of the present invention.

[0125] 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 aspects, the antibody comprises a mutation that increases dissociation of the antibody or an antigen-binding portion thereof from sCD28 at low pH. In some embodiments, the low pH is a pH of 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 or less. Each possibility represents a separate embodiment of the invention. In some embodiments, the low pH is pH 6 or less. In some embodiments, the low pH is the pH found in human endosomes. In some embodiments, the low pH is the pH found in human lysosomes. In some aspects, the antibody comprises a mutation that increases dissociation of the antibody or an antigen-binding portion thereof from sCD28 at low calcium concentration. In some embodiments, the low calcium concentration is a calcium concentration of 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 or less. Each possibility represents a separate embodiment of the 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 the CDR. In some embodiments, the antibody comprises a mutation that increases dissociation of the antibody from sCD28 in endosomes and / or lysosomes.

[0126] 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 mutations to histidine at 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. In some embodiments, the mutation increases the uptake of the antibody and its bound antigen into cells.

[0127] Examples of mutations that are useful for increasing Fc receptor binding, increasing cellular uptake, increasing dissociation at low pH, and / or generating a sweeping antibody are well known in the art. Such examples can be found, for example, in Schroter et al., A generic approach to engineer antibody pH-switches using combinatorial histidine scanning 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, all of which are incorporated herein by reference in their entirety. Methods for generating such antibodies and methods for testing their efficacy are also described therein. In some embodiments, the mutations are those of the human Fc domain and are selected from mutations from histidine 268 to glutamine, valine 309 to leucine, alanine 330 to serine, and proline 331 to serine. The numbering provided is for IgG1, but equivalent mutations as may be determined by one of ordinary skill in the art can be made in other IgGs. Furthermore, the efficacy of such antibodies can be examined by performing binding / dissociation assays in media of various pHs. Testing in model organisms such as mice and monkeys is also possible, and the serum levels of sCD28 are measured before and after addition of the antibody. Optionally, tumors expressing human sCD28 may be xenografted into the animals to ensure serum sCD28.

[0128] In some embodiments, the antibody comprises mutations in the CDRs that reduce binding to the 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 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 invention. In some embodiments, amino acids outside the CDRs are mutated to histidine.

[0129] The incorporation of histidine residues into the binding interface of the antibody has been shown to be used to engineer pH-dependent binding. The basis for pH-sensitive binding results from the sensitivity of histidine to protonation as a result of the decrease in pH value in various microenvironments, including the intracellular microenvironment, more specifically endosomal vesicles, and more preferably early endosomes. Protonation of the side chain of histidine at the binding interface can change electrostatic interactions or induce conformational changes leading to differences in the pH-dependence of binding affinity.

[0130] The incorporation of pH sensitivity into the antigen-binding site can increase the number of antigen-binding cycles, and the pH-dependent antibody binds to the antigen with a similarly high or reduced sufficient affinity at plasma pH (pH 7.4), shows a decrease in binding at low pH, resulting in a faster and increased dissociation of the antibody from its antigen-binding site within acidic endosomes, thereby allowing for increased recirculation to the plasma and clearance of the antigen from the plasma.

[0131] His-scanning, which replaces each amino acid of a CDR with histidine and evaluates pH-dependent binding, is a well-established technique known in the art. Multiple histidine mutations can be combined to increase the effect of the mutations synergistically or linearly. Combinatorial his-scanning library techniques using phage or yeast display are also well known in the art.

[0132] 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.

[0133] 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 mimic. 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 conjugate protein. In some embodiments, the synthetic peptide is based on a non-antibody scaffold. In some embodiments, the agent is an antibody mimic. In some embodiments, the antibody mimic has a molar mass of less than 100, 90, 80, 70, 60, 50, 40, 30 or 20 kDa. Each possibility represents a separate embodiment of the invention. In some embodiments, the agent is a nucleic acid aptamer. In some embodiments, the aptamer is DNA. In some embodiments, the aptamer is RNA. In some embodiments, the aptamer is DNA or RNA.

[0134] In some embodiments, the antibody is for use in the treatment and / or prevention of cancer in a subject in need thereof. In some embodiments, the antibody is for use in improving immunotherapy in a subject in need thereof. In some embodiments, the immunotherapy is immunotherapy based on PD1 and / or PD-L1. PD-1 and PD-L1 immunotherapies are well known in the art and include blockade of PD-1 and PD-L1, as well as PD-1 and PD-L1 inhibitors. Some examples include, but are not limited to, pembrolizumab (Keytruda), nivolumab (Opdivo), pidilizumab, semiprimab, atezolizumab (Tecentriq), avelumab (Bevancio), and durvalumab (Imfinzi).

[0135] Blocking of mCD28 shedding In another aspect, an agent that binds to membrane CD28 (mCD28) and inhibits proteolytic cleavage of mCD28 is provided.

[0136] As used herein, inhibition of proteolytic cleavage refers to any reduction in the proteolytic cleavage of mCD28. In some embodiments, the inhibition is a reduction in cleavage of 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 invention. In some embodiments, inhibition of proteolytic cleavage maintains the level of mCD28 on immune cells. In some embodiments, inhibition of proteolytic cleavage increases the level of mCD28 on immune cells. In some embodiments, inhibition of proteolytic cleavage maintains a level of mCD28 appropriate for immune stimulation.

[0137] In some embodiments, the reduction of proteolytic cleavage is a reduction of cleavage by at least one protease. In some embodiments, the reduction of proteolytic cleavage is a reduction of cleavage by at least one metalloprotease. In some embodiments, the metalloprotease is ADAM10, ADAM17, or both.

[0138] In some embodiments, the agent is an antibody or an antigen-binding fragment thereof. 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 particularly increases the half-life in serum. In some embodiments, the half-life extending protein is human serum albumin. In some embodiments, the agent is modified by a chemical substance that generates a modification that improves the half-life. In some embodiments, the modification is pegylation and the chemical substance is polyethylene glycol. It is understood by those skilled in the art that any half-life extending protein or chemical substance, or modification known in the art can be used.

[0139] 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 a 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 binds only to a single molecule of CD28. In some embodiments, the agent binds to monomeric CD28. In some embodiments, the agent binds to dimeric CD28. In some embodiments, the agent binds to monomeric and / or dimeric CD28.

[0140] In some embodiments, the agent is not a CD28 agonist. In some embodiments, the agent is not a CD28 antagonist. In some embodiments, the agent is neither a CD28 agonist nor an antagonist.

[0141] In some embodiments, the agent does not bind to the ligand-binding domain of mCD28. In some embodiments, the agent does not shield or block access to the ligand-binding domain. In some embodiments, the agent binds to the cleavage site. In some embodiments, the agent shields or blocks access to the cleavage site. In some embodiments, the agent blocks access to the cleavage site of the protease. In some embodiments, the agent binds to the stalk region of CD28. In some embodiments, the agent binds to the membrane-proximal region of mCD28. In some embodiments, the cleavage site is within the stalk region. In some embodiments, the stalk region comprises the sequence GKHLCPSPLFPGPSKP (SEQ ID NO: 9). In some embodiments, the stalk region comprises the sequence KGKHLCPSPLFPGPS (SEQ ID NO: 36). In some embodiments, the stalk region comprises or consists of the sequence HVKGKHLCPSPLFPGPSKP (SEQ ID NO: 10).

[0142] 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 invention. In some embodiments, the cleavage site is before leucine 145 of SEQ ID NO: 1.

[0143] In some embodiments, the agent does not modulate the function and / or signaling of CD28. In some embodiments, the agent does not degrade mCD28. In some embodiments, the agent does not lead to or promote mCD28 degradation. In some embodiments, the signaling is mCD28-mediated immune cell activation. In some embodiments, the agent does not inhibit the activation of immune cells. In some embodiments, the agent does not induce internalization or recycling of the CD28 receptor. Co-stimulation via mCD28 is essential for T cell immune activation. By proteolytic cleavage, the ligand-binding domain in the extracellular region of CD28 is removed from the transmembrane and cytoplasmic portions of the protein remaining in the membrane. Thus, the cleaved CD28 cannot signal and cannot contribute to T cell activation. Thus, an agent that blocks cleavage and is also an antagonist does not allow activation of mCD28. Similarly, an agent that blocks cleavage but is also an agonist may induce abnormal T cell activation and potentially an autoimmune response. 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.

[0144] 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 invention.

[0145] In some embodiments, the binding of the agent to the cell does not kill the cell. In some embodiments, the binding of the agent to the cell does not lead to cell death. In some embodiments, the agent does not induce antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the agent does not induce complement-dependent cytotoxicity (CDC). In some embodiments, the agent does not induce ADCC and / or CDC. In some embodiments, the agent is an antibody and comprises an IgG2 or IgG4 domain. In some embodiments, the antibody comprises an IgG2 domain. In some embodiments, the antibody comprises an IgG4 domain. In some embodiments, the antibody comprises an IgG1 or IgG3 that has been mutated to reduce cell death mediated by antibody binding. In some embodiments, the mutation mutates the Fc receptor binding domain. In some embodiments, the Fc domain of the antibody is engineered or mutated to reduce CDC, ADCC, or both. Fc engineering is well known in the art and any mutation or amino acid change known to reduce antibody-mediated cell killing can be used.

[0146] In some embodiments, the agent is a non-antibody protein. In some embodiments, the agent is a small molecule. In some embodiments, the agent is a nucleic acid molecule. In some embodiments, the agent is a synthetic peptide. In some embodiments, the agent is a synthetic binding protein. In some embodiments, the synthetic peptide is based on a non-antibody scaffold. In some embodiments, the agent is an antibody mimetic. In some embodiments, the antibody mimetic has a molar mass of less than 100, 90, 80, 70, 60, 50, 40, 30 or 20 kDa. Each possibility represents a separate embodiment of the invention. In some embodiments, the agent is a nucleic acid aptamer. In some embodiments, the aptamer is DNA. In some embodiments, the aptamer is RNA. In some embodiments, the aptamer is DNA or RNA. Examples of antibody mimetics include, but are not limited to, affilins, affimers, affitins, alphabodies, anticalins, avimers, DARPins, finomers, Kunitz domain peptides, monobodies, and nanoCLAMPs. In some embodiments, the antibody mimetic is a DARPin.

[0147] In some embodiments, the agent inhibits proteolytic cleavage by at least one protease. In some embodiments, the protease is a metalloprotease. In some embodiments, the protease is a matrix metalloprotease. In some embodiments, the protease is a serine protease. In some embodiments, the protease is a cysteine protease. In some embodiments, the protease is a threonine protease. In some embodiments, the protease is a serine, cysteine or threonine protease. In some embodiments, the protease is an aspartic protease. In some embodiments, the protease is a glutamic protease. In some embodiments, the protease is selected from aspartic, glutamic, serine, cysteine and threonine proteases. In some embodiments, the protease is an asparaginyl peptidase. In some embodiments, the protease is a sheddase. In some embodiments, the metalloprotease is an exopeptidase. In some embodiments, the metalloprotease is an endopeptidase. In some embodiments, the metalloprotease is an exopeptidase or an endopeptidase. In some embodiments, the metalloprotease is a zinc catalyst. In some embodiments, the metalloprotease is a cobalt catalyst. In some embodiments, the metalloprotease is ADAM10. In some embodiments, the metalloprotease is ADAM17. In some embodiments, the metalloprotease is ADAM10 and / or ADAM17. In some embodiments, the metalloprotease is ADAM10, ADAM17, or both.

[0148] 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).

[0149] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence DVKLVESGGGLVKLGGSLKLSCVASGFTFSSYYMSWVRQTPEKRLEWVATISDGGDNTYYAGTVTGRFTISRDFAKNTLYLQMNSLTSEDTAVYYCARIHWPYYFDSWGQGTTLTVSS (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 aspects, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence GACGTGAAGCTCGTGGAGTCTGGGGGAGGCTTAGTGAAGCTTGGAGGGTCCCTGAAACTCTCCTGTGTAGCCTCTGGATTCACTTTCAGTAGCTATTACATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCGACCATAAGTGATGGTGGTGATAACACCTACTACGCAGGCACTGTGACGGGCCGATTCACCATCTCCAGAGACTTTGCCAAGAACACCCTGTACCTGCAAATGAACAGTCTGACCTCTGAGGACACAGCCGTGTATTACTGTGCAAGAATTCATTGGCCTTACTATTTTGACTCCTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO: 54). In some embodiments, the heavy chain consists of SEQ ID NO: 54. The anti-cleavage antibody of the present application has been sequenced and found to have a heavy chain consisting of SEQ ID NO: 54. The CDRs of this heavy chain determined using the Chothia scheme are SEQ ID NOs: 30-32.

[0150] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the 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 aspects, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence CAATTTGTTCTCTCCCAGTCTCCAGCAATCCTGTCTGCATCTCCCGGGGAGATGCTCACAATGACTTGCAGGGCCAGCTCAAGTGTAAGTTATATGAACTGGTATCAGCAGAAGCCAGGATCTTCCCCCAAACCCTGGATTTATGCCACATCCGACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTATTCTCTCACAATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTCACCCACCCACGTTCGGAGGGGGGACCAAGCTGGAAATAAGA (SEQ ID NO: 56). In some embodiments, the light chain consists of SEQ ID NO: 56. The anti-cleavage antibody referred to in this application was found to have a light chain that was sequenced and consists of SEQ ID NO: 56. The CDRs of this light chain determined using the Chothia scheme are SEQ ID NOs: 33-35.

[0151] Method of treating / preventing cancer In another aspect, there is provided a method of treating and / or preventing cancer in a subject in need thereof, the method comprising reducing the soluble CD28 (sCD28) level in said subject.

[0152] In another aspect, there is provided a method of improving immunotherapy in a subject in need thereof, the method comprising reducing the sCD28 level in said subject.

[0153] In some embodiments, the immunotherapy is immunotherapy based on PD-1 and / or PD-L1. In some embodiments, the PD-1 / PD-L1 based immunotherapy includes administering an anti-PD1 or anti-PD-L1 antibody. In some embodiments, the therapy includes blocking the PD-1 checkpoint. In some embodiments, the immunotherapy includes administering allogeneic, syngeneic, or autologous immune cells to a subject. In some embodiments, the immune cells are T cells. In some embodiments, the subject in need of immunotherapy has cancer.

[0154] As used herein, the term "treating" or "treatment" of a disease, disorder, or condition encompasses alleviating at least one symptom thereof, reducing its severity, or inhibiting its progression. Treatment need not mean that the disease, disorder, or condition is completely cured. For a treatment to be effective, a useful composition herein need only reduce the severity of the disease, disorder, or condition, reduce the severity of the symptoms associated therewith, or provide an improvement in the quality of life of the patient or subject.

[0155] In some embodiments, reducing comprises administering to the subject at least one agent of the present invention. As used herein, terms such as "administering", "administration", etc. refer to any method of delivering a composition comprising an active agent to a subject in a manner that provides a therapeutic effect in sound medical practice. One aspect of the subject matter provides oral administration of a therapeutically effective amount of an agent of the present invention to a patient in need thereof. Other suitable routes of administration may include parenteral, subcutaneous, intravenous, intramuscular, or intraperitoneal.

[0156] In another aspect, there is provided a pharmaceutical composition comprising an agent of the present invention and a therapeutically acceptable carrier, adjuvant or excipient. In some embodiments, administering comprises administering the pharmaceutical composition of the present invention.

[0157] As used herein, the terms "carrier," "excipient," or "adjuvant" refer to any component of a pharmaceutical composition that is not an active drug. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material, any type of formulation aid, or simply a sterile aqueous medium such as physiological saline. Some examples of materials that can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol and phosphate buffer solutions, and other non-toxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of substances that can serve 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 physiological saline, phosphate buffer solutions, cocoa butter (suppository base), emulsifying agents, and other non-toxic pharmaceutically compatible substances used in other pharmaceutical formulations. Wetting agents and lubricants such as sodium lauryl sulfate, and coloring agents, flavoring agents, excipients, stabilizers, antioxidants, and preservatives may also be present. For formulating the compositions contemplated herein, non-toxic, inert and effective carriers can be used.In this regard, suitable pharmaceutically acceptable carriers, excipients, and diluents are well known to those skilled in the art, for example, as described in The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, N.J. (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and the "Inactive Ingredient Guide", U.S. Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, the entire contents of all of which are hereby incorporated by reference in their entirety. Examples of pharmaceutically acceptable excipients, carriers, and diluents useful in the present compositions include distilled water, physiological saline, Ringer's solution, dextrose solution, Hank's solution, and DMSO. These additional inactive ingredients, as well as effective formulations and administration procedures, are well known in the art and are described in standard textbooks such as Goodman and Gillman’s: The Pharmacological Bases of Therapeutics, 8th Ed., Gilman et al., Eds., Pergamon Press (1990); Remington’s Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005), the entire contents of each of which are hereby incorporated by reference in their entirety.The compositions described herein may also be included in artificially produced structures such as liposomes, ISCOMs, sustained release particles, and other vehicles that increase the half-life of peptides or polypeptides in serum. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. Liposomes for use with the peptides described herein are formed from standard vesicle-forming lipids that generally include neutral and negatively charged phospholipids and sterols such as cholesterol. The choice of lipids is generally determined by considerations such as the size of the liposome and its stability in the blood. For example, as discussed by Coligan, J.E. et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, various methods are available for preparing liposomes, see also U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0158] The carrier may generally constitute from about 0.1% to about 99.99999% by weight of the pharmaceutical composition presented herein.

[0159] In some embodiments, reducing comprises administering an inhibitory nucleic acid molecule that binds to the mRNA encoding sCD28 and does not bind to the mRNA encoding mCD28. One possible source of sCD28 is from the translation of a transcriptional variant of CD28 lacking a transmembrane domain. Such a variant may have a unique mRNA sequence that can be targeted to degrade or inhibit the translation of the mRNA. As a non-limiting example, sCD28 is produced by an mRNA splice variant lacking exon 3 (SEQ ID NO: 7). In this variant, the exon 2 and exon 4 junction is the only sequence present in the variant but lacking full-length CD28. In some embodiments, the nucleic acid molecule binds to at least the junction of exon 2 and exon 4 of SEQ ID NO: 7. In some embodiments, the splice junction of exons 2 and 4 comprises 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.

[0160] In some embodiments, the nucleic acid molecule is siRNA. In some embodiments, the molecule is shRNA. In some embodiments, the molecule is siRNA or shRNA.

[0161] In some embodiments, reducing comprises administering an agent that inhibits a protease capable of cleaving mCD28. In some embodiments, the agent inhibits ADAM10, ADAM17, 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. Additional therapeutic protease inhibitors are disclosed in International Patent Application WO2004096139.

[0162] In some embodiments, reducing comprises administering a peptide comprising the stalk region of CD28 or a fragment thereof. In some embodiments, the peptide is a monomer. In some embodiments, the peptide is a dimer. In some embodiments, CD28 is human CD28. In some embodiments, the peptide inhibits access to a protease cleavage site. In some embodiments, the peptide induces the production of autoantibodies that block the cleavage site.

[0163] In some embodiments, the method of the invention does not degrade or lead to the degradation of mCD28. In some embodiments, the method of the invention does not reduce the level of mCD28 on immune cells. In some embodiments, the method of the invention does not reduce mCD28-mediated immune cell activation. In some embodiments, the method of the invention maintains the level of mCD28 on the immune cells of a subject. In some embodiments, the method of the invention increases the level of mCD28 on the immune cells of a subject.

[0164] In some embodiments, the reduction is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% reduction of sCD28. Each possibility represents a separate embodiment of the invention. In some embodiments, the reduction is a reduction of serum sCD28. In some embodiments, the reduction is a reduction of the blood level of sCD28. In some embodiments, the reduction is a reduction of the sCD28 level in the tumor microenvironment (TME).

[0165] In some embodiments, the subject's blood contains high levels of sCD28. In some embodiments, the subject's blood before the decrease contains high levels of sCD28. In some embodiments, the level is elevated compared to that of a healthy subject. In some embodiments, the subject's sCD28 level is 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% higher than that of a healthy subject. Each possibility represents a separate embodiment of the invention. In some embodiments, the level is elevated above 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45, or 50 ng / ml of blood. Each possibility represents a separate embodiment of the invention. In some embodiments, the level is elevated above 5 ng / ml. In some embodiments, the level is elevated above 10 ng / ml. In some embodiments, the level is elevated above 20 ng / ml. In some embodiments, the subject's blood contains at least 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45, or 50 ng of sCD28 per ml of blood. Each possibility represents a separate embodiment of the invention. In some embodiments, the subject's blood before the decrease contains at least 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45, or 50 ng of sCD28 per ml of blood. Each possibility represents a separate embodiment of the invention. In some embodiments, the subject's blood contains at least 5 ng / ml of sCD28. In some embodiments, the subject's blood contains at least 10 ng / ml of sCD28. In some embodiments, the subject's blood contains at least 20 ng / ml of sCD28. In some embodiments, the subject's blood before the decrease contains at least 5 ng / ml of sCD28. In some embodiments, the subject's blood before the decrease contains at least 10 ng / ml of sCD28.In some embodiments, the subject's blood before reduction contains at least 20 ng / ml of sCD28.

[0166] 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 is receiving PD-1 / PD-L1 therapy. In some embodiments, the subject is a non-responder to PD-1 / PD-L1 therapy. In some embodiments, the subject is naive to PD-1 / PD-L1 therapy. In some embodiments, the method of the present invention is performed together with PD-1 / PD-L1 therapy. In some embodiments, the method of the present invention is performed before PD-1 / PD-L1 therapy.

[0167] 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 CAR-T. In some embodiments, the CAR is CAR-NK. In some embodiments, the another immunotherapy is a cancer vaccine.

[0168] As used herein, the terms "CAR-T cells" and "CAR-NK cells" refer to engineered receptors that are specific for at least one target protein (e.g., an immunogenic protein whose expression is increased after treatment with an epigenetic modifier) and are grafted onto immune effector cells (T cells or NK cells). In some embodiments, the CAR-T cells have the specificity of a monoclonal antibody grafted onto T cells. In some embodiments, the CAR-NK cells have the specificity of a monoclonal antibody grafted onto NK cells. In some embodiments, the T cells are selected from cytotoxic T lymphocytes and regulatory T cells.

[0169] CAR-T and CAR-NK cells and their vectors are well known in the art. Such cells target the protein to which the receptor binds and exhibit cytotoxicity. In some embodiments, the CAR-T or CAR-NK cells target at least one viral protein. In some embodiments, the CAR-T or CAR-NK cells target multiple viral proteins. In some embodiments, the CAR-T or CAR-NK cells target a viral protein whose expression is increased by contact with an epigenetic modifier.

[0170] The construction of CAR-T cells is well-known in the art. In one non-limiting example, monoclonal antibodies against viral proteins can be produced, and then vectors encoding the antibodies are constructed. The vectors also include co-stimulatory signal regions. In some embodiments, the co-stimulatory signal region includes the intracellular domain of a known T cell or NK cell stimulatory molecule. In some embodiments, the intracellular domain is selected from at least one of CD3Z, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind 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.

[0171] In some embodiments, the cancer is a cancer with elevated sCD28 levels. In some embodiments, the cancer comprises high sCD28 levels. In some embodiments, the elevated and / or high sCD28 levels are levels of 5, 6, 7, 8, 9, 10, 12, 14, 15, 17, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 ng / ml and / or levels exceeding that. Each possibility represents a separate embodiment of the 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% of the levels in a healthy subject and / or levels exceeding that. Each possibility represents a separate embodiment of the 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, and colorectal cancer. In some embodiments, the cancer is 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 invention.

[0172] In some embodiments, the method is performed in vivo. In some embodiments, the method is performed in vitro. In some embodiments, the reducing is performed in vivo. In some embodiments, the reducing is performed in vitro. In some embodiments, the reducing comprises removing blood from a subject, reducing the sCD28 levels in the removed blood, and returning the blood to the subject, thereby reducing sCD28 in the subject. Methods of dialysis and hemofiltration are well known. The present invention may be performed by sweeping sCD28 in vitro and then returning the blood to the subject.

[0173] Method of Use In another aspect, a method for detecting sCD28 in a sample in vitro, comprising: a. providing a sample containing sCD28; b. contacting the sample with an agent of the present invention; and c. detecting the agent bound to sCD28; thereby providing a method for detecting sCD28 in the sample.

[0174] 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 cells. In some embodiments, the detection is by a secondary antibody. In some embodiments, the detection is a labeling molecule that binds to the agent. In some embodiments, the detection is by ELISA. In some embodiments, the detection is by immunohistochemistry. In some embodiments, the detection is by immunoblot. In some embodiments, the agent of the present invention is for detecting sCD28. In some embodiments, the agent of the present invention is for detecting sCD28 without detecting mCD28.

[0175] In another aspect, a method for determining the suitability of a subject to be treated by the treatment method of the present invention, comprising providing a sample from the subject and determining the sCD28 level in the sample, wherein an increase in the sCD28 level indicates that the subject is suitable to be treated by the treatment method of the present invention.

[0176] 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 immunotherapy based on CD80 and / or CD86, the method comprising providing a sample from the subject and determining the sCD28 level in the sample, wherein an sCD28 level above 5 ng / ml indicates that the subject is unsuitable to be treated by anti-PD-1 and / or PD-L1 immunotherapy or immunotherapy based on CD80 and / or CD86 is provided.

[0177] In another aspect, a method for creating an inappropriate subject suitable for receiving anti-PD-1 and / or PD-L1 immunotherapy and / or immunotherapy based on CD80 and / or CD86, the method comprising reducing the sCD28 levels in the inappropriate subjects, thereby rendering them suitable is provided.

[0178] In another aspect, a method for improving immunotherapy based on CD80 and / or CD86 in a subject, the method comprising: a. measuring the sCD28 level of the subject; and b. increasing the dosage of immunotherapy based on CD80 and / or CD86 to a subject having an sCD28 level above 5 ng / ml thereby providing a method for improving immunotherapy based on CD80 and / or CD86.

[0179] In some embodiments, the method further comprises rendering an inappropriate subject suitable by performing the method of the invention.

[0180] In some embodiments, the elevated level rises to near the level in a healthy subject. In some embodiments, the elevated level rises to near the level of a predetermined threshold. In some embodiments, the elevated level is an sCD28 level that exceeds 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 ng / ml. Each possibility represents a separate embodiment of the invention. In some embodiments, an sCD28 level that exceeds 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 ng / ml indicates that the subject is unsuitable for treatment. Each possibility represents a separate embodiment of the invention. In some embodiments, an sCD28 level that exceeds 10 indicates that the subject is unsuitable for treatment. In some embodiments, an sCD28 level that exceeds 20 indicates that the subject is unsuitable for treatment.

[0181] CD80 and CD86 immunotherapies are well known in the art and involve administering CD80 / CD86 and / or mimetics, derivatives or analogs thereof to stimulate an immune response. CD80-Fc, as a non-limiting example, is currently in clinical trials as an anti-cancer immunotherapeutic agent.

[0182] In some embodiments, detecting an agent bound to sCD28 involves 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 for determining the suitability of a subject treated with a pharmaceutical composition of the invention. In some embodiments, an sCD28 level that exceeds a predetermined threshold indicates that the subject is suitable for treatment with the composition of the invention. In some embodiments, the method is for determining the suitability of a subject for immunotherapy. In some embodiments, an sCD28 level that is below a predetermined threshold, or the absence of sCD28, indicates that the subject is suitable for immunotherapy. In some embodiments, an sCD28 level that exceeds a predetermined threshold indicates that the subject is suitable for combination use of immunotherapy and the composition of the invention.

[0183] In some embodiments, the subject has cancer. In some embodiments, the subject is at risk of developing cancer.

[0184] 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 exceeds a predetermined threshold. In some embodiments, the method further comprises subjecting the subject to immunotherapy.

[0185] Kit In another aspect, there is provided a kit comprising at least one agent of the invention or a pharmaceutical composition of the invention.

[0186] In some embodiments, the kit further comprises an immunotherapeutic agent based on PD-1 and / or PD-L1. In some embodiments, the kit comprises a label indicating that the agent of the invention is for use in combination with an immunotherapeutic agent based on PD-1 and / or PD-L1. In some embodiments, the kit comprises a label indicating that a therapeutic agent based on PD-1 and / or PD-L1 is for use in combination with the antibody or pharmaceutical composition of the invention.

[0187] In some embodiments, the kit further comprises a detection molecule for detecting the agent of the invention. In some embodiments, the detection molecule is a secondary detection molecule. In some embodiments, the detection molecule binds to the agent. Detection molecules are well known in the art and include, but are not limited to, fluorescent moieties and molecules, dyes, and secondary antibodies.

[0188] In another aspect, there is provided a kit comprising an immunotherapeutic agent based on PD-1 and / or PD-L1 and comprising a label indicating that it is for use in combination with the antibody or pharmaceutical composition of the invention.

[0189] In some embodiments, the kits of the invention are for use in treating cancer. In some embodiments, the kits of the invention are diagnostic kits. In some embodiments, the kits of the invention are for use in determining the serum level of sCD28 in a subject in need thereof. In some embodiments, the subject has cancer. In some embodiments, the kits of the invention are for use in determining the suitability of a subject to be treated with the agents or pharmaceutical compositions of the invention. In some embodiments, the kits are for use in determining the suitability of a subject to be treated with anti-PD-1 / PD-L1 based immunotherapy.

[0190] Method for generating an agent In another aspect, a method for generating an agent of the invention, comprising: a. obtaining an agent that specifically binds to the CD28 extracellular domain or a fragment thereof; and b. 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; Thereby providing a method for generating an agent of the invention.

[0191] In another aspect, a method for generating an agent of the invention, comprising: culturing a host cell comprising one or more vectors comprising a nucleic acid sequence encoding an agent, the nucleic acid sequence being i. for obtaining an agent that binds to the CD28 extracellular domain or a fragment thereof; ii. testing the ability of the agent to block the cleavage of mCD28 by a protease; and iii. selecting at least one agent that blocks the cleavage of mCD28 by a protease wherein the agent is selected by culturing, thereby providing a method for generating an agent of the invention.

[0192] In some embodiments, the agent is an anti-cleavage agent. In some embodiments, the agent is an anti-shedding agent. In some embodiments, the agent reduces shedding of sCD28 in a subject. In some embodiments, the agent reduces cleavage of mCD28. In some embodiments, the agent reduces cleavage of mCD28 in a subject.

[0193] In some embodiments, the protease is ADAM10. In some embodiments, the protease is ADAM17. In some embodiments, the protease is ADAM10, ADAM17, or both.

[0194] As used herein, the term "extracellular domain of CD28" refers to the N-terminal portion of CD28 that comes 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 stalk domain. In some embodiments, the extracellular domain of CD28 comprises the stalk domain of CD28. In some embodiments, the extracellular domain of CD28 comprises, or consists of, the sequence NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 37). In some embodiments, the extracellular domain of CD28 or a fragment thereof is a dimer. In some aspects, the extracellular domain of CD28 or a fragment thereof is a monomer. In some aspects, the extracellular domain of CD28 or a fragment thereof is a dimer or a monomer.

[0195] As used herein, "fragment" refers to a partial polypeptide that constitutes part of a larger protein or protein domain. In some embodiments, the fragment comprises at least 10, 20, 30, 40, or 50 amino acids. Each possibility represents a separate embodiment of the invention. In some embodiments, the fragment comprises up to 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids. Each possibility represents a separate embodiment of the invention. In some embodiments, obtaining an agent that binds to a fragment of the extracellular domain of CD28 is obtaining an agent that specifically binds to the CD28 stalk domain.

[0196] In some embodiments, the method further comprises assaying mCD28 downstream signaling in the presence of the obtained agent and selecting at least one agent that does not substantially activate or antagonize mCD28 signaling. In some embodiments, selecting is selecting at least one agent that does not antagonize mCD28 signaling. It will be understood by those skilled in the art that while stimulating CD28 signaling for the treatment of cancer may not be harmful, antagonizing the signaling may have the opposite effect.

[0197] In some embodiments, testing the ability of an agent to block cleavage is by the method described below. In some embodiments, testing the ability of an agent to block cleavage involves mixing the agent, a protease, and the extracellular domain of CD28 or a fragment thereof containing the cleavage site. In some embodiments, testing further comprises sequencing the extracellular domain of CD28 or a fragment thereof to check for truncation and / or cleavage. In some embodiments, testing further comprises electrophoresing the extracellular domain of CD28 or a fragment thereof on a gel having sufficient sensitivity to measure size changes due to cleavage. In some embodiments, testing further comprises measuring the production of sCD28 from cells expressing mCD28 in the presence of the agent and the protease.

[0198] In another aspect, a method for generating a medicament of the present invention, comprising: a. obtaining a medicament that binds to the CD28 extracellular domain or a fragment thereof; and b. assaying mCD28 downstream signaling in the presence of the obtained medicament and selecting at least one medicament that neither substantially activates nor substantially antagonizes mCD28 signaling; Thereby providing a method for generating a medicament of the present invention.

[0199] In another aspect, a method for generating a medicament of the present invention, comprising: culturing a host cell containing one or more vectors comprising a nucleic acid sequence encoding a medicament, wherein the nucleic acid sequence is i. obtained from a medicament that binds to the CD28 extracellular domain or a fragment thereof; ii. assaying mCD28 downstream signaling in the presence of the obtained medicament; and iii. selected from at least one medicament that neither substantially activates nor substantially antagonizes mCD28 signaling Thereby providing a method for generating a medicament of the present invention.

[0200] In some embodiments, the medicament is a scavenger. In some embodiments, the medicament is for removing sCD28 from a subject. In some embodiments, the medicament specifically binds to CD28. In some embodiments, the medicament specifically binds to sCD28.

[0201] In some embodiments, the method further comprises isolating and / or extracting the medicament from the host cell. In some embodiments, the method further comprises isolating and / or extracting the medicament from the culture medium of the host cell. In some embodiments, the method further comprises purifying the medicament from the host cell or the culture medium of the host cell.

[0202] In some embodiments, the method further comprises testing the binding of the obtained agent to mCD28 and selecting at least one agent that does not bind to mCD28. In some embodiments, the method further comprises testing the binding of the obtained agent to sCD28 from a subject and selecting at least one agent that binds to sCD28 from the subject. In some embodiments, the subject is human. In some embodiments, the subject is an autoimmune patient.

[0203] In some embodiments, obtaining an agent comprises immunizing an organism with the 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, camels, chickens, goats, and phages. In some embodiments, the camel is selected from camels and llamas. In some embodiments, collecting comprises blood collection. In some embodiments, collecting comprises a. extracting B cells from the spleen of the immunized organism; b. fusing the extracted B cells with myeloma cells to generate hybridomas; and c. collecting antibodies from the hybridomas.

[0204] In some embodiments, obtaining an agent comprises screening a library of agents for binding to the CD28 extracellular domain or a fragment thereof, and selecting an agent that binds as such. In some embodiments, the library is a phage display library. In some embodiments, the library is an immunized 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 chains, single domains or nanobodies. In some embodiments, obtaining an agent comprises sequencing the agent. In some embodiments, obtaining an agent comprises generating a recombinant form of the agent. In some embodiments, selecting an agent comprises sequencing the agent. In some embodiments, selecting an agent comprises generating a recombinant form of the agent. In some embodiments, the recombinant form is generated from the sequence of the agent. In some embodiments, the method further comprises humanizing the agent.

[0205] Expressing a nucleic acid molecule encoding an agent intracellularly is well known to those skilled in the art. It can be carried out, among many methods, by transfection, viral infection, or direct alteration of the cell's genome. In some embodiments, the gene is in an expression vector such as a plasmid or viral vector. One such example of an expression vector containing p16-Ink4a is the mammalian expression vector pCMV p16 INK4A available from Addgene.

[0206] Vector nucleic acid sequences generally include at least an origin of replication for growth intracellularly, and optionally, a heterologous polynucleotide sequence, expression control elements (e.g., a promoter, an enhancer), a selectable marker (e.g., antibiotic resistance), a polyadenylation sequence.

[0207] The vector may be a DNA plasmid delivered by a non-viral method or a viral method. The viral vector may be a retroviral vector, a herpes virus vector, an adenovirus vector, an adeno-associated virus vector or a poxvirus vector. The promoter may be active in mammalian cells. The promoter may be a viral promoter.

[0208] In some embodiments, the nucleic acid sequence encoding the agent is operably linked to a promoter. The term "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that enables expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or, when the vector is introduced into a host cell, in the host cell).

[0209] In some embodiments, the vector is introduced into cells by standard methods including electroporation (e.g., as described in From et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985)), heat shock, infection with a viral vector, high-velocity ballistic penetration by small particles with nucleic acids within or on the surface of small beads or particles (Klein et al., Nature 327, 70-73 (1987)), etc.

[0210] As used herein, the term "promoter" refers to a group of transcriptional control modules clustered around the start site of RNA polymerase, i.e., RNA polymerase II. The promoter consists of individual functional modules, each of which consists of DNA of approximately 7-20 bp and contains one or more recognition sites for transcriptional activator or repressor proteins.

[0211] 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 as well as most snRNA and microRNA.

[0212] In some embodiments, mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(±), pGL3, pZeoSV2(±), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81, (available from Invitrogen), pCI (available from Promega), pMbac, pPbac, pBK-RSV and pBK-CMV (available from Strategene), pTRES (available from Clontech), and their derivatives.

[0213] In some embodiments, expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses are used according to the present invention. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papillomavirus include pBV-1MTHA, and vectors derived from Epstein-Barr virus include pHEBO and p2O5. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and any other vector that enables the expression of a protein under the direction of the SV-40 early promoter, SV-40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or any other promoter shown to be effective for expression in eukaryotic cells.

[0214] In some embodiments, recombinant viral vectors that provide advantages such as horizontal infection and target specificity are used for in vivo expression. In one embodiment, horizontal infection is, for example, inherent in the retroviral life cycle, a process by which a single infected cell produces many progeny virions that bud and infect adjacent cells. In one embodiment, as a result, a large area that was mostly initially uninfected by the original virus particles becomes rapidly infected. In one embodiment, viral vectors that cannot spread horizontally are produced. In one embodiment, this feature can be useful when the desired goal is to introduce a particular gene into only a localized number of target cells.

[0215] A variety of methods can be used to introduce the expression vectors of the 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 at. [Biotechniques 4(6):504-512, 1986], and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. Further, for positive-negative selection methods, see U.S. Patent Nos. 5,464,764 and 5,487,992.

[0216] In addition to containing the elements necessary for transcription and translation of the inserted coding sequence (encoding a polypeptide), it is understood that the expression constructs of the present invention may also include sequences engineered to optimize the stability, production, purification, yield, or activity of the expressed polypeptide.

[0217] In another aspect, there is provided an agent produced by the method of the present invention.

[0218] In another aspect, there is provided a pharmaceutical composition comprising an agent produced by the method of the present invention, and a pharmaceutically acceptable carrier, excipient, or adjuvant.

[0219] As used herein, the term "about" when combined with a value refers to plus and minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm ± 100 nm.

[0220] It should be noted that as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides, and reference to "a polypeptide" includes reference to one or more polypeptides and their equivalents known to those skilled in the art. Further, it should be noted that the claims may be drafted to exclude any element. Thus, this description is intended to serve as a precedent for the use of exclusive terms such as "solely", "only", etc. in connection with the recitation of claim elements or the use of "negative" limitations.

[0221] In cases where a convention similar to “at least one of A, B, and C, etc.” is used, generally, such a construction is intended in the sense that one of ordinary skill in the art would understand the convention (for example, “a system having at least one of A, B, and C” would include, but not be limited to, a system having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by one of ordinary skill in the art that in any of the specification, claims, or drawings, any substantially discrete words and / or phrases presenting two or more alternative terms are to be understood as contemplating the possibility of including one of the terms, any of the terms, or any of the terms. For example, the phrase “A or B” would be understood to include the possibilities of “A” or “B” or “A and B”.

[0222] It is understood that certain features of the invention that are described in the context of separate embodiments for clarity can also be provided in combination in a single embodiment. Conversely, various features of the invention that are described in relation to a single embodiment for brevity can also be provided separately or in any suitable partial combination. All combinations of embodiments related to the invention are specifically encompassed by the invention and are disclosed herein as if each and every combination were individually and explicitly disclosed. Further, all partial combinations of various embodiments and their elements are also specifically encompassed by the invention and are disclosed herein as if each and every such partial combination were individually and explicitly disclosed herein.

[0223] Additional objects, advantages, and novel features of the invention will become apparent to those skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the invention described above and claimed in the following claims section of the specification is experimentally verified in the following examples.

[0224] With respect to the various embodiments and aspects of the invention described above and claimed in the following claims, experimental verification is recognized in the following examples.

Example

[0225] 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 well explained in the literature. For example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R.M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); U.S. Patent Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; the methodologies described in "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J.E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J.E., ed. (1994); Stites et al.(Eds.), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (Eds.), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996) are hereby incorporated by reference in their entirety. Other general references are described throughout this specification.

[0226] Materials and Methods Antibodies - Commercially available mouse monoclonal anti-CD28 clone #CD28.2 (Biolegend, catalog number 302902) and FITC-conjugated (Biolegend, catalog number 302906). Goat polyclonal anti-CD28 (R&D 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 immune research, catalog number 709-605-149). Goat anti-mouse IgG HRP (Jackson immune research, catalog number 115-035-071). Anti-human CD3 clone OKT3 (Biolegend, catalog number 317304). Anti-human PD-1 pembrolizumab (MK-3475). Human IgG (Sigma, catalog number I4506).

[0227] To facilitate affinity purification, generation of recombinant soluble human CD28a - CD28a cDNA was subcloned into the pCDNA3.1 vector having a c - terminal TEV protease cleavage site in front of six histidine residues. HEK293 cells were transfected using the plasmid, and soluble recombinant CD28a was purified by immobilized metal affinity chromatography (IMAC). The pooled material was subjected to His - tag cleavage using TEV protease after the second IMAC to remove the His - tag and TEV protease.

[0228] ELISA - The amounts of human interferon - γ (Biolegend, catalog number 430103), human interleukin 2 (Biolegend, catalog number 431802), human interleukin 6 (Biolegend, catalog number 430502), human interleukin 10 (Biolegend, catalog number 430603), human tumor growth factor β1 (Biolegend, catalog number 436708), human interleukin β1 (Biolegend, catalog number 437004) and human CD28 (R&D system, catalog number DY342) were quantified using commercially available ELISA kits. Cell proliferation and viability (MTT assay) were performed according to the manufacturer's (Roche, catalog number 11465007001) instructions. The kynurenine (IDO activity) ELISA kit was performed according to the manufacturer's (ImmuSmo catalog number BA E - 2200) instructions.

[0229] Cytokine multiplex - Simultaneous evaluation of several cytokines was performed using ProcartaPLex (Invitrogen, catalog number PPX - 07 - MXXGPY2) on a Magpix system (Millipore).

[0230] Flow cytometry - Generally, cells were kept on ice at all steps. Before staining, 5x10 5Cells were blocked with 50 μg / mL human IgG (Sigma, catalog number I4506) in FACS buffer (PBS containing 0.1% BSA) for 15 minutes. Antibodies were used at the concentrations recommended by the manufacturer and incubated in the dark for 30 minutes. Incubation was performed in a 96-well U-bottom plate with a volume of 100 μL. Cells were washed twice with 200 μL of FACS buffer and transferred to FACS tubes in 150 μL of FACS buffer for analysis. Cells were analyzed on a Gallios Flow Cytometer (Beckman Coulter) using Kaluza of Gallios Flow Cytometry Acquisition Software.

[0231] Isolation of cell lines and human immune cells - The Jurkat leukemic T cell lymphoblast 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). CD3 cells were isolated from fresh blood samples of healthy donors by negative selection using the RosetteSEP™ Human T Cell Enrichment Kit (STEMCELL, catalog number 15061). CD4 cells were isolated from fresh blood samples of healthy donors by negative selection using the EasySep™ Human CD4 T Cell Enrichment Kit (STEMCELL, catalog number 19059). Monocytes were isolated from fresh blood samples of healthy donors by negative selection using the EasySep™ Human Monocyte Enrichment Kit (STEMCELL, catalog number 17952). All cells were grown in complete RPMI-1640 medium supplemented with 10% HI-FCS and a pen / strep mixture.

[0232] Differentiation of dendritic cells - Monocytes were cultured in RPMI medium containing growth factors refreshed on days 3 and 6 at a density of 1 × 10 6Cultured at a density of / mL. Immature dendritic cells (iDCs) were induced for 6 days with 50 ng / mL GM-CSF and 20 ng / mL IL-4. If necessary, iDCs were further differentiated into mature dendritic cells by adding 100 ng / mL LPS for 48 hours. The generated cell population was tested for the indicated phenotypes by FACS analysis of relevant markers and analysis of the secretion of characteristic cytokines.

[0233] Protease inhibitors - Protease inhibitors were added at the concentrations indicated at the start of each experiment. In the 1-week assay, additional inhibitor was added at the final concentration 3 days later. 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). The protease cocktail, when mentioned, was a mixture of TAPI-1 and GM6001 in an equimolar ratio.

[0234] PHA activation of CD4 T cells or Jurkat T cell lines for the production of soluble CD28 - 1×10 5 Jurkat cells or CD4 T cells were incubated for an additional 5 days (Jurkat) or 7 days (CD4 T cells) with the indicated concentrations of phytohemagglutinin (Sigma, catalog number L8902) and various protease inhibitors.

[0235] SEB or CMV activation of PBMCs for the production of soluble CD28 - In 48-well plates, 0.3×10 6 PBMCs were stimulated with 0.5 ng / mL SEB (Sigma, catalog number S4881) at 37°C for 5 - 7 days with or without various protease inhibitors at the indicated concentrations. Alternatively, in a 96-well plate format assay, 0.1×10 6 PBMCs were stimulated with 0.5 ng / mL SEB. For CMV stimulation, in 96-well plates, 0.5×10 6PBMCs were stimulated with 0.5 μg / mL CMV peptivator (Milteny Biotec, catalog number 130-093-435) at 37 °C for 2 - 5 days with various protease inhibitors at the indicated concentrations, either present or absent. In the continuous depletion experiment, PBMCs were stimulated with SEB or CMV in 24-well plates for 24 hours, the cells were harvested, washed three times with RPMI without stimulant, and re-seeded in 96-well plates. Samples were taken at the indicated times and placed under freezing conditions until the assay for soluble CD28.

[0236] Mixed lymphocyte reaction - 1×10 5 of immature DCs were mixed with 5×10 5 of isolated autologous CD3 T cells for 6 days.

[0237] SEB or CMV stimulation assay using ectopically recombinant human CD28, human CTLA-4 and human CD80 - In the CMV stimulation, in 96-well plates, 0.5×10 6 PBMCs (from healthy or cancer patient donors) were stimulated with 0.5 μg / mL CMV peptivator (Milteny Biotec, catalog number 130-093-435) at 37 °C for 2 - 5 days with recombinant human CD28 (R&D systems, catalog number 342-CD), human CTLA-4 (R&D systems, catalog number 434-CT), human CD80 (R&D systems, catalog number 140-B1) at the indicated concentrations, either present or absent. In the SEB setting, 1×10 5 PBMCs were cultured for 72 hours at a concentration of 0.5 ng / mL of staphylococcal enterotoxin B (SEB) (Sigma, catalog number S4881) in the presence of the indicated concentrations of rec. human CD28. When specified, anti-PD1 or human IgG was added at a final concentration of 5 μg / mL.

[0238] Autologous monocyte CD3 MLR - 0.5×10 6 T cells from the same CMV-reactive donor were mixed with 0.5×10 5 monocytes and stimulated with 0.5 μg / mL CMV peptivator at 37 °C for 6 days with the indicated concentrations of treatment, either present or absent.

[0239] Stimulation of monocytes by recombinant human CD28 - 1.5×10 6 Monocytes of were seeded in 24-well plates for 48 hours in RPMI medium containing 100 - 100 U / ml of IFNγ (R&D system, catalog number 285-IF) in the presence of recombinant human CD28 at the indicated concentrations. The generated cell population was tested for the indicated phenotypes by FACS analysis of related markers (IDO, PD-L1, and PD-L2) and analysis of the secretion of a characteristic cytokine (IL-6).

[0240] T cell stimulation by OKT3 - 0.1×10 6 Isolated CD3 T cells (from healthy donors) were stimulated with the indicated amount of anti-CD3 clone OKT3 at 37 °C for 48 - 72 hours. Recombinant human CD80-Fc (2 μg / mL, R&D system) was added in soluble form when described. An antibody against CD28 or a control was added at the indicated concentrations in soluble form.

[0241] CD86 blockade FACS - 0.5×10 of HEK293 cells stably transfected with human CD28 were incubated with 5 μg / mL of CD86-Fc (R&D systems, catalog number 141-B2) for 30 minutes at room temperature with or without an anti-CD28 antibody (20 μg / mL). The cells were washed and harvested for secondary binding using anti-human heavy and light chain antibodies conjugated to fluorophores at a 1:5000 dilution for 20 minutes on ice. 6

[0242] Co-culture of SCC-25 cancer cell line and monocytes in a transwell-based assay - With or without the indicated treatment for 4 days in serum-free starvation medium, 4×10 of SCC-25 cells were seeded on the bottom of 24-well plates, and 1×10 4 5 Monocytes were seeded on cell culture inserts (Millipore, catalog number MCHT241148).

[0243] Identification, cloning, and sequencing of CD28 mRNA variants - Human PBMCs were stimulated with 0.5 ng / mL SEB (Sigma, catalog number S4881) at 37°C for 7 days. Human CD4 T cells were incubated with phytohemagglutinin (2 μg / mL, Sigma, catalog number L8902) at 37°C for 7 days. Total RNA was extracted from the cell pellets of activated and naive immune cells using the RNeasy Mini Kit (Qiagen, catalog number 74106) with the Qiacube automated system (Qiagen). From each sample, 500 ng of RNA was subjected to RT reaction using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, catalog number 4374966). Negative controls for the RT reaction were tubes without RNA and another tube without reverse transcriptase. 1 μL of cDNA was subjected to PCR using the forward primer (CD28F) 5’-ATGCTGAGGCTGCTCTTGGCTCTCAAC-3’ (SEQ ID NO: 38) and the reverse primer (CD28R) 5’-TCAGGAGCGATAGGCTGCGAAGTCGCG-3’ (SEQ ID NO: 39). The PCR product was loaded onto a 1% agarose gel. The PCR product was excised from the gel and extracted using the QIAquick Gel Extraction Kit. When indicated, Sanger sequencing was performed using the CD28F primer.

[0244] Detection of soluble human CD28 in the plasma of cancer patients - Twenty frozen plasma samples each from 10 cancer indications and healthy donors were purchased from DxBiosamples (San Diego, CA, USA). Plasma samples were diluted 1:20 and analyzed for soluble human CD28 by ELISA. Samples with high sCD28 were re-analyzed at appropriate dilutions.

[0245] Direct CD28 EIA - Unless otherwise specified, Corning high - binding plates or equivalent were used for screening. 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 a BSA - conjugated dimer peptide composed of the CD28 stalk region amino acid sequence (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 antibody under investigation after detection with goat anti - mouse HRP Fc - specific 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.

[0246] Simulation of depletion of soluble CD28 from plasma of cancer patients. Antibodies or recombinant proteins were coated onto tosyl - activated magnetic beads (Invitrogen, catalog number DY - 14203) as described in the manufacturer's protocol. Beads were harvested to represent the indicated amount of antibody and mixed with plasma samples from cancer patients. The mixture was incubated in a thermomixer at 37°C at 1000 RPM for 2 hours, followed by removal of the beads using a DynaMag magnet (Invitrogen, catalog number 12321D), and the samples were assayed using a CD28 - specific ELISA.

[0247] Antibody Sequencing. For amino acid sequencing, the antibody was provided to Rapid Novor. Sequencing was performed using standard methods, which briefly included LC-MS analysis after enzymatic digestion with six enzymes (pepsin, trypsin, chymotrypsin, elastase, Lys C, and Asp N). Digestion was performed using disulfide reduction and alkylation. LC-MS / MS analysis was performed using a Thermo-Fisher Q-exactive mass spectrometer. For both the heavy and light chains of each antibody, 100% of the amino acid residues were covered by at least five peptide scans and contained significant supporting fragment ions. CDRs were determined using the Chothia scheme.

[0248] Example 1: Human CD28 Undergoes Proteolytic Shedding during Chronic Stimulation In the culture of chronically stimulated human PBMCs, soluble CD28 (sCD28) was detected by ELISA (Figure 1). This phenomenon was evident regardless of the nature of the stimulant, artificial (SEB) or physiological (CMV), indicating the robustness of the phenomenon. Treatment with TAPI-1 and GM6001 (broad-spectrum MMP and ADAM17 inhibitors) decreased the amount of sCD28 in a dose-dependent manner, suggesting that the origin of soluble CD28 is due to shedding from the membrane form (Figure 1). The cellular source of the shed CD28 is T cells, as shown in Figure 2. Chronic stimulation of the Jurkat T cell line or human CD4 T cells from the peripheral blood of healthy donors with PHA produced sCD28 in a dose-dependent manner (Figure 2, upper panel). Treatment with TAPI-1 and GM6001 decreased the amount of sCD28 in a dose-dependent manner at each PHA concentration (Figure 2, upper panel) and at a fixed PHA concentration (Figure 2, lower graph).

[0249] Treatment with the highly specific ADAM-10 inhibitor GI254023X resulted in dose-dependent near-complete inhibition of sCD28 release from activated immune cells (Figure 3A, lower panel). Similar results were observed with the ADAM-17 specific inhibitor TMI-1 (Figure 3B, lower panel). The viability of immune cells was monitored by MTT assay to confirm the metabolic activity of the cells in culture. The results showed no significant difference between treatments with and without the ADAM inhibitor, suggesting that the low sCD28 levels were due to protease activity blockade and not an artifact of cell death caused by protease inhibitors (Figure 3A - B, upper panel).

[0250] The generation of sCD28 was also verified in a more physiological system. First, CD4 T cells were utilized that mimic the physiological stimulation of T cells by isolated autologous dendritic cells and antigen-presenting cells. The increase in sCD28 became evident upon mixing the two cell populations and was further enhanced when CMV was added to the culture (Figure 4A). This indicates that upon chronic stimulation, the human CD28 protein undergoes a proteolytic shedding process.

[0251] Next, human PBMCs were stimulated with CMV peptides (Figure 4B) or SEB (Figure 4C) for 24 hours. The cells were then washed to remove the stimulant and re-seeded without the stimulatory signal for various periods. Subsequently, the presence of sCD28 in the medium was examined. The accumulation of sCD28 was clearly visible over time. Furthermore, the accumulation was dependent on the activity of ADAM-10 and ADAM-17 as seen in Figure 4D. When specific inhibitors were added at different concentrations after SEB stimulation, the amount of sCD28 quantified after 120 hours decreased. Since CD28 shedding occurs after primary activation of T cells and does not necessarily require constant or repeated stimulation, this study can explain the presence of large amounts of soluble CD28 in the blood of patients.

[0252] The final evidence that the origin of sCD28 is due to proteolytic shedding was obtained from the observation that a known alternatively spliced variant of CD28 is significantly downregulated in activated lymphocytes (Figure 5). Four CD28 mRNA products could be detected in unstimulated samples, but only two were apparent in activated cells. The top band was confirmed by Sanger sequencing to correspond to full-length mature CD28 that is membrane-bound upon translation, while the second band (black arrow, ~500 bp) was previously shown to be an alternatively spliced product that gives rise to a secreted truncated protein. This band from the naive sequence was confirmed by Sanger sequencing to be a splice variant lacking exon 3 and thus the transmembrane domain. Stimulation of PBMC and T cells with SEB or PHA preferentially expressed full-length CD28 mRNA and concomitantly suppressed the spliced transcript (Figure 5, lanes 5 and 7). Collectively, these results indicate that the origin of sCD28 from activated T cells is proteolytic shedding and not alternative splicing at the gene level.

[0253] Example 2: Soluble human CD28 has immunosuppressive activity As seen in Figure 1, reducing the level of sCD28 using a cocktail of protease inhibitors directly correlates with an increase in T cell activation, as evident by the level of secreted IFNγ, suggesting that sCD28 has an immunosuppressive function. Increasing the concentration of the protease inhibitor cocktail led to a decrease in sCD28 levels in the cell culture medium, and these decreases in sCD28 levels were inversely correlated with an increase in the level of secreted IFNγ. To further investigate immunosuppression by sCD28, recombinant human CD28 lacking the transmembrane and cytoplasmic domains was added to the culture of human PBMC 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, supporting the robustness of this signaling axis blocked by sCD28.

[0254] In parallel, an increase in interleukin-6 secretion (Figs. 7 and 8A) and interleukin-10 (Fig. 8A) was evident. These cytokines have been reported to indicate a skewing of the immune system towards a type 2 immune response that can assist cancer growth and angiogenesis via suppression of immune effector activity (IL-10), as well as STAT-3 signaling (IL-6). Furthermore, a comparison was made with soluble CTLA-4 (mimicking abatacept - a registered therapeutic for autoimmune disorders), and a generally similar effect on the immune system was revealed with respect to the cytokine secretion profile (Fig. 8A).

[0255] Next, human PBMCs were stimulated with SEB (1 ng / mL) in the presence or absence of recombinant human CD28. Human IgG was used as a control. Lymphocyte clustering, a prominent feature of immune activation, was monitored using IncuCyte® S3 live cells in photographs taken every 12 hours. As seen in Fig. 8C, SEB essentially had no effect on lymphocytes when recombinant human sCD28 was present. During in-vitro immune responses, antigen-presenting cells (APCs) cluster with each other and with other cell types, and it is well established that clustering is essential for antigen-specific activation of resting lymphocytes. Soluble CD28 appears to decrease the amount and size of cluster formation during the SEB immune response, which means inhibiting the first step of T cell-specific activation by APCs.

[0256] Similar results were observed when isolated autologous monocytes and CD3 T cells were co-cultured in a mixed lymphocyte reaction (MLR). The mixed cells were stimulated with CMV peptide (0.5 μg / mL) for 5 days with or without increasing the concentration of recombinant human sCD28. Again, sCD28 was found to inhibit IFNγ secretion while increasing the secretion of IL-1B, TGFβ and IL-10 (Fig. 8B).

[0257] sCD28 had a similar immunosuppressive effect on monocytes. The enzyme indoleamine 2,3-dioxygenase (IDO) is involved in immune regulation by virtue of its ability to catabolize the essential amino acid tryptophan. It is also expressed in various immune cells and many cancer cells. Tryptophan deficiency inhibits the maturation and proliferation of T lymphocytes, while kynurenine, the end product of tryptophan catabolism, is also known as an immunosuppressive metabolite that promotes immune tolerance in various physiological and pathophysiological states. To test the effect of sCD28 on IDO, isolated human monocytes were stimulated with IFNγ (1000 U / mL) for 48 hours in the presence of control human IgG or recombinant human CD28 (10 μg / mL). After incubation, the monocytes were intracellularly stained with human IDO (Figure 8E). To facilitate intracellular staining, the cells were fixed and permeabilized with the BD Cytofix / Cytoperm buffer kit. The culture supernatants of various treatments were evaluated for IDO activity using the ImmuSmol proprietary kynurenine ELISA kit (Figure 8D). sCD28 strongly enhanced IDO expression in monocytes.

[0258] Furthermore, surprisingly, sCD28 was found to be a potent inhibitor of anti-PD1 immunotherapy. MK-3475 (pembrolizumab or Keytruda, Merck) is an approved drug with unprecedented efficacy in multiple cancer indications. Addition to PMBC cultures increased inflammatory cytokine secretion (IFNγ and IL-2), but the presence of sCD28 completely abrogated this immune activation effect (Figure 9A).

[0259] Similar results were observed in the MLR setting. The MLR was performed as before with and without sCD28, and with and without the anti-PD1 antibody (MK3475, 5 μg / mL) (Figure 9B). As expected, MK-3475 increased IFNγ secretion and decreased TGFβ secretion. In particular, in the presence of sCD28, the effect of MK-3475 was significantly reduced.

[0260] To elucidate the mechanism by which sCD28 inhibits the pro-activation effect of anti-PD-1 therapy, the expression of PD-1 ligand on immune cells was examined in the presence of sCD28. Isolated human monocytes were stimulated with IFNγ (1000 U / mL) or recombinant human CD28 (10 μg / mL) for 48 hours in the presence of control human IgG (10 μg / mL). After incubation, the monocytes were stained with PD-L1 (Figure 9C, left) and PD-L2 (Figure 9C, right). Both ligands were upregulated on monocytes cultured with sCD28, suggesting one possible way by which sCD28 circumvents the effect of anti-PD-1 immunotherapy.

[0261] Example 3: Soluble human CD28 is found in the plasma of cancer patients The levels of sCD28 in cancer have only been shown in a small number of breast cancer patients and were found to be only slightly above the levels observed in healthy individuals (Isitmangil, G., In vivo, 2016). The authors suggest that sCD28 could be used as a marker for breast cancer, but no functional relationship has been suggested. Now that it has been found that soluble CD28 may actually enhance cancer evasion of the immune system, an investigation of 220 samples covering 10 different cancer indications and 20 samples from healthy donors was conducted. In the investigation, high sCD28 levels were found in some cancers, but these levels could be orders of magnitude higher than those seen in healthy controls or breast cancer patients (Figure 10A). In fact, the levels in breast cancer patients appear to be equivalent to those in healthy individuals when compared to the sCD28 levels seen in some melanoma, colorectal cancer, ovarian cancer, NSCLC, and head and neck cancer patients. This investigation was conducted using antibodies #1 and #3 of the present invention. It was surprising that such high levels had not been previously reported 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 a commercially available CD28 kit (catalog number DY342) available from R&D Systems. The antibodies of the present invention detected very high levels of sCD28 in 3 of the samples and lower levels in another 14, while the commercially available kit did not detect sCD28 in any of the samples (Figure 10B). Similar results were also found with a commercially available CD28 ELISA kit BMS290 available from Thermo Fischer Scientific, which was used in the study by Isitmangil et al. regarding breast cancer. The inability of commercially available kits to detect sCD28 shed from human cells could explain why the high sCD28 in cancer patients has been an unknown phenomenon until now.

[0262] To further elucidate the role of sCD28 in cancer, PBMCs were isolated from cancer patients with different indications. Cells were stimulated with SEB (5 ng / mL) for 3 days alone, with MK-3475, with recombinant human sCD28, or with a combination of both molecules. In the presence of MK-3475, the concentration of human IFNγ in the supernatant of cells from all donors was significantly reduced in the presence of sCD28 (Figure 10C). Indeed, the effect of MK-3475 was absent due to sCD28.

[0263] Next, cells of the head and neck cancer cell line SCC-25 were incubated alone or with monocytes in a transwell assay. When IL-6 was administered as a positive control to SCC-25 cells grown alone, cell proliferation measured by MTT (Figure 10D, top) and by confluence% (Figure 10D, bottom) actually increased. Growth of cancer cells in the presence of monocytes also increased, but the greatest increase was decidedly observed when sCD28 was included in the co-culture. This data further supports that sCD28 has a pre-cancer effect.

[0264] Example 4: sCD28 inhibits the efficacy of CD80-Fc CD80 is one of the two major ligands of mCD28, together with CD86. The extracellular domain of CD80 fused to the Fc portion has been used as an immune-stimulatory molecule and investigated 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 of soluble recombinant human CD80-Fc. As expected, CD80-Fc increased IFNγ secretion. However, the addition of sCD28 abrogated the secondary activation effect of CD80-Fc (Figure 11A). Similarly, when isolated PBMCs were stimulated with CMV peptide for 3 days and then incubated with sCD28, it was necessary to increase the amount of CD80-Fc to generate the expected immune response (Figure 11B).

[0265] Example 5: Effect of sCD28 on cancer in vivo Since mice do not cleave mCD28, the effect of sCD28 cannot be easily examined in a mouse model. The closest option is to administer recombinant sCD28 to mice to mimic the situation of elevated sCD28 levels. This was investigated in the H22 syngeneic mouse model. Balb / c fully immunocompetent mice were xenografted with H22 hepatocellular carcinoma cells. The cells grew even in fully immunocompetent mice, and the addition of anti-PD-1 therapy almost completely stopped tumor growth (Figure 12A). When recombinant human sCD28 was added, the effect of anti-PD-1 therapy became almost completely ineffective in two mice (Figure 12B). This suggests that in some subjects, an increase in sCD28 levels may have a very harmful effect on cancer progression.

[0266] Example 6: Characterization of an antibody-based agent that eliminates the immunosuppressive effect (anti-cleavage) of sCD28 The discovery that human CD28 undergoes proteolytic processes by ADAM10 and ADAM17 prompted an examination of the polypeptide sequences of its candidate regions that exhibit potential susceptibility to proteolytic shedding. Studies have suggested that ADAM10 and ADAM17 prefer leucine, valine, and aromatic residues at the P1’ position. The most intriguing sequence region of human CD28 is the stalk section spanning from histidine 134 to proline 152 (SEQ ID NO: 10: HVKGKHLCPSPLFPGPSKP), which connects the globular IgV domain to the transmembrane region. This region holds a total of three leucine and valine residues, as well as a phenylalanine residue, and is predicted to have no secondary structure elements that could impede protease access. In particular, the stalk region also contains cysteine 141, which forms a disulfide bond that promotes the homodimerization of CD28. CD1 mice were immunized with a dimeric peptide that mimics the CD28 stalk region, for the purpose of generating an antibody or antibody fragment that specifically binds to the CD28 stalk region and potentially blocks the access of different proteases to shed CD28 while avoiding the reduction of the structure and function of CD28 oligomers. The peptide sequence used for immunization was SEQ ID NO: 40, GKHLCPSPLFPGPSKPK, and a C-terminal lysine was added to have a free amino group that enables KLH or BSA conjugation using hydrazide chemistry. The conjugation was performed between the hydrazide-terminated CD28 peptide and S-4FB-modified BSA, which generates a free aldehyde for site-specific conjugation. Dimerization was confirmed by running the peptide on a non-denaturing gel.

[0267] Six mice were immunized with the BSA-conjugated peptide. Mouse tail blood sera were routinely checked by ELISA for binding to the target antigen, recombinant human CD28 (rhCD28), and the KLH-conjugated mixed peptide.

[0268] For the initial clone screening to bind to the antigen target by ELISA, a high-throughput screening platform was used. Culture supernatants were used as primary antibodies to generate and screen approximately 2000 hybridomas in an initial EIA against rhCD28, and color development was carried out using HRP-conjugated goat anti-mouse IgG(γ) and TMB. Twenty clones were obtained, all of which were IgG2a antibodies and were further expanded for purification and isotyping. In the ELISA assay, it has been confirmed that all 20 IgGs have various levels of significant binding to recombinant soluble CD28 (Table 1).

Table 1

[0269] All three of the highest-binding clones were sequenced and found to be the same antibody (hereinafter referred to as clone M9), so experiments were continued using this antibody. The sequence of antibody M9 can be found above. Serial dilutions of antibody M9 were used to confirm specific binding to recombinant human sCD28 and stalk region peptides (Figure 13A). Interestingly, the antibody was able to detect recombinant human sCD28 but not sCD28 that had actually shed from immune cells (Figure 13B). This strongly suggests that the antibody binds at the cleavage site and that the deisotope to which it binds is incomplete in the cleaved form. Direct inhibition of cleavage was checked by mixing rhCD28 with ADAM10 or ADAM17 in the presence and absence of antibody M9. The resulting rhCD28 peptides were sequenced by mass spectrometry to determine whether cleavage had occurred.

[0270] These results suggest that the association of the stalk region may not directly affect mCD28 signaling. However, it has been reported that the monoclonal antibody MAB342, clone 37407 from R&D Systems binds to the stalk region of CD28 (see International Patent Application WO2004096139). Furthermore, R&D Systems has 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 investigated. MAB342 was found to bind to recombinant human CD28-Fc and recombinant human CD28a (a soluble splice variant lacking the stalk region sequence), but not to recombinant mouse CD28 (Figure 13C). However, when assaying 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 (Figure 13C).

[0271] Example 7: Characterization of agents based on antibodies that eliminate (sweep away) the immunosuppressive effect of sCD28 Three antibodies that strongly bind to sCD28 were obtained and their potential effectiveness as sweep agents to lower sCD28 levels was investigated. The sequences of the three antibodies are shown above.

[0272] To confirm the ability of the 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 Figure 14A. Similarly, when human PBMC were stimulated with SEB, the antibodies of the present invention were again able to strongly detect sCD28 (Figure 14B). Using the protease inhibitors TAPI, GI254023X, and TMI-1, lower levels of sCD28 were detected, confirming that sCD28 was indeed being detected.

[0273] Antibodies #1 and #2 were compared to the control commercial CD28 antibody CD28.2, which is known to bind to mCD28. Direct ELISA was performed using various concentrations of sCD28 and each antibody (and mIgG as a negative control) (Figure 14C). Recombinant human sCD28 protein was immobilized on maxisorp ELISA plates and binding was evaluated by detection of the bound antibody with donkey anti-mouse IgG (H&L)-HRP and color development with TMB. The commercial antibody CD28.2 bound to sCD28, but the binding was poor and the increase in O.D. was only slight even when the input was increased 10-fold. In contrast, both antibodies #1 and #2 showed much stronger binding to sCD28, with antibody #1 showing a detection almost 10-fold greater and antibody #2 showing a linear relationship with increasing antibody concentration over the entire range tested. The direct ELISA for each antibody is shown individually in Figure 14D. Interestingly, antibody #2 was also able to bind to mouse CD28, while antibodies #1 and #3 were not able to bind (Figure 14E).

[0274] CD28.2 is known to stimulate T cell proliferation and cytokine secretion and thus acts as an mCD28 agonist. Indeed, when the binding of CD86 to mCD28 was measured by FACS, addition of CD28.2 resulted in a significant decrease in CD86 binding (Figure 15A), indicating that CD28.2 binds to or occludes the ligand-binding domain of mCD28. In contrast, neither antibody #1 (Figure 15B), antibody #2 (Figure 15C), nor antibody #3 (Figure 15D) blocked the binding of CD86 to mCD28, and in fact, both antibodies appear to correspond to the mIgG control (Figure 15E).

[0275] As a representative of inflammatory cytokine secretion by T cells, interferon γ (IFNγ) secretion was measured. In the presence of anti-CD3 stimulation, the antibody CD28.2 induced strong IFNγ secretion indicating activation of T cells. In contrast, antibodies #1, #2, and #3 all had no effect on IFNγ secretion at various concentrations (Figure 15F). Therefore, CD28.2 acts as an mCD28 agonist, while antibodies #1-3 are not agonist-like. Similar results were also seen when human PBMCs were stimulated with SEB (Figure 15G).

[0276] Similarly, when human isolated T cells are stimulated with anti-CD3 antibody, CD80-Fc functions as an agonist and increases IFNγ secretion. Addition of an antagonist should decrease the effect of CD80, but when antibodies #1-3 were added, no decrease in secretion was observed (Figure 15H). This indicates that antibodies #1-3 are not antagonist-like either.

[0277] Finally, the ability of antibodies #1-3 to bind to mCD28 was examined. It is not essential but advantageous for an sCD28 antibody not to bind to mCD28. Such an antibody can be used for specific detection of soluble proteins and, when used therapeutically, will not affect the membrane form. Naïve isolated CD3 T cells were evaluated by FAC to assay mCD28 binding. The cells were incubated with antibodies #1-3 (dark gray histograms at a concentration of 20 μg / mL) or isotype control (mIgG, 20 μg / mL, light gray histograms). Binding detection was performed using an Alexa Fluor 647-conjugated goat anti-mouse secondary antibody. As seen in Figure 15I, only antibody #1 bound to mCD28. Antibodies #2 and #3 are completely specific for sCD28 as they do not bind to CD28 on the cell surface.

[0278] Example 8: sCD28 can be removed from plasma using an antibody According to the manufacturer's protocol (Thermo Fisher Scientific), various amounts (0.125 - 2 μg) of antibody #1 and #2 were added to tosyl-activated magnetic beads. Subsequently, the added beads were added to plasma samples from cancer patients, and the mixture was incubated at 37°C for 2 hours at 1000 RPM using a thermomixer. The beads were separated and removed from the plasma using a magnet, and the amount of sCD28 remaining in the serum was measured by ELISA. Representative results from antibody #1 (Figure 16A) and antibody #2 (Figure 16B) are shown, indicating that the antibodies of the present invention were able to bind and remove sCD28 from the plasma.

[0279] Antibody #1 was further tested in various cancer samples (melanoma, colorectal cancer, and ovarian cancer) and compared with three B7 molecules that are known ligands of CD28. Approximately 1.5 μg of antibody #1, CD80, CD86, and ICOS-L were each added to tosyl-activated magnetic beads, and the beads were mixed with plasma as before. Colorectal cancer samples (Figure 16C), two melanoma samples (Figures 16D - E), ovarian cancer samples (Figure 16F), and samples from healthy donors (Figure 16G) were all tested. Antibody #1 was superior to the other three molecules tested and was able to reduce sCD28 levels in the plasma to levels below those observed in healthy samples in all cases. In fact, antibody #1 reduced sCD28 to almost undetectable levels in all samples except one melanoma sample (Figure 16D), and even in this sample, a reduction of more than 50% was observed.

[0280] The present invention has been described in conjunction with its specific embodiments, but it is obvious that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

Claims

**Claim 1** An antibody or an antigen-binding fragment thereof that binds to the stalk region of membrane CD28 (mCD28) and inhibits proteolytic cleavage of said mCD28, wherein the stalk region consists of the amino acid sequence HVKGKHLCPSPLFPGPSKP (SEQ ID NO: 10), said antibody or antigen-binding fragment thereof comprises three heavy-chain CDRs (CDR-H) and three light-chain CDRs (CDR-L), CDR-H1 comprises the amino acid sequence shown in SEQ ID NO: 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), an antibody or antigen-binding fragment thereof. **Claim 2** a. A heavy chain comprising the amino acid sequence of SEQ ID NO: 53; and b. A light chain comprising the amino acid sequence of SEQ ID NO: 55 The antibody or antigen-binding fragment thereof according to claim 1, comprising at least one of them. **Claim 3** A heavy chain comprising a heavy-chain variable region consisting of the amino acid sequence of SEQ ID NO: 53 and a light chain comprising a light-chain variable region consisting of the amino acid sequence of SEQ ID NO: 55, the antibody or antigen-binding fragment thereof according to claim 2. **Claim 4** An antibody or an antigen-binding fragment thereof that binds to the stalk region of membrane CD28 (mCD28) and inhibits proteolytic cleavage of said mCD28, which competes with an antibody comprising a heavy chain comprising a heavy-chain variable region consisting of the amino acid sequence of SEQ ID NO: 53 and a light chain comprising a light-chain variable region consisting of the amino acid sequence of SEQ ID NO: 55, and wherein the stalk region consists of the amino acid sequence of SEQ ID NO: 10, an antibody or antigen-binding fragment thereof. **Claim 5** The antibody or antigen-binding fragment thereof is a. Neither a CD28 agonist nor an antagonist, b. Neither degrades said mCD28 nor inhibits mCD28-mediated immune cell activation, c. Does not induce antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), d. Inhibits proteolytic cleavage by at least one protease, and optionally, said at least one protease is a metalloprotease. e. which is an antibody, f. which comprises IgG2 or IgG4, g. which is a Fab fragment, h. which is a single-chain antibody, or i. which is a combination thereof, The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4. **Claim 6** An antibody or antigen-binding fragment thereof that binds to soluble CD28 (sCD28), does not bind to membrane CD28 (mCD28), and is neither a CD28 agonist nor an antagonist, comprising three heavy-chain CDRs (CDR-H) and three light-chain CDRs (CDR-L), CDR-H1 comprises the amino acid sequence shown in SEQ ID NO: 18 (GYTFT SY), CDR-H2 comprises the amino acid sequence shown in SEQ ID NO: 19 (YPGDG D), CDR-H3 comprises the amino acid sequence shown in SEQ ID NO: 20 (NYRYSS FGY), CDR-L1 comprises the amino acid sequence shown in SEQ ID NO: 21 (KSSQSL LN SGNQK NYLT), CDR-L2 comprises the amino acid sequence shown in SEQ ID NO: 22 (WASTRE S), CDR-L3 comprises the amino acid sequence shown in SEQ ID NO: 23 (QSDYSY PLT); or CDR-H1 comprises the amino acid sequence shown in SEQ ID NO: 24 (GYTFTD Y), 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 (SSPYYD SNHFDY), CDR-L1 comprises the amino acid sequence shown in SEQ ID NO: 27 (SARS SI NYMH), CDR-L2 comprises the amino acid sequence shown in SEQ ID NO: 28 (DTSKLA S), CDR-L3 comprises the amino acid sequence shown in SEQ ID NO: 29 (HQRNSY PFT), the antibody or antigen-binding fragment thereof. **Claim 7** a. a heavy chain comprising the amino acid sequence of SEQ ID NO: 45; and b. a light chain comprising the amino acid sequence of SEQ ID NO: 47 The antibody or antigen-binding fragment thereof according to claim 6, comprising at least one of them. **Claim 8** The antibody or antigen-binding fragment thereof according to claim 7, comprising a heavy chain comprising a heavy-chain variable region consisting of the amino acid sequence of SEQ ID NO: 45 and a light chain comprising a light-chain variable region consisting of the amino acid sequence of SEQ ID NO:

47. **Claim 9** a. a heavy chain comprising the amino acid sequence of SEQ ID NO: 49; and b. a light chain comprising the amino acid sequence of SEQ ID NO: 51 The antibody or antigen-binding fragment thereof according to claim 6, comprising at least one of them. **Claim 10** The antibody or antigen-binding fragment thereof according to claim 9, comprising a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 49 and a light chain comprising a light chain variable region consisting of the amino acid sequence of SEQ ID NO:

51.

11. An antibody or antigen-binding fragment thereof that binds to soluble CD28 (sCD28) with higher affinity than membrane CD28 (mCD28) and is neither a CD28 agonist nor an antagonist, 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).

12. a. A heavy chain comprising the amino acid sequence of SEQ ID NO: 41; and b. A light chain comprising the amino acid sequence of SEQ ID NO: 43 The antibody or antigen-binding fragment thereof according to claim 11, comprising at least one of.

13. The antibody or antigen-binding fragment thereof according to claim 12, comprising a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 41 and a light chain comprising a light chain variable region consisting of the amino acid sequence of SEQ ID NO:

43.

14. The antibody or antigen-binding fragment thereof a. An antibody, Fab fragment, Fv, Fab, F(ab'), ２ , scFv, scFv ２ fragment, humanized antibody, and single-chain antibody selected from b. Does not inhibit the binding of the sCD28 to its ligand, c. Binds to dimeric sCD28, monomeric sCD28, or both, d. Binds outside the IgV domain of sCD28, e. The binding to sCD28 in a living organism results in at least one of the degradation of the bound sCD28, the removal of the bound sCD28 from the blood, and the transport of the bound sCD28 to lysosomes, endosomes, proteasomes, or combinations thereof, f. Does not induce antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), or g. Combinations thereof, The antibody or antigen-binding fragment thereof according to any one of claims 6 to 13.

15. An antibody or antigen-binding fragment thereof for use in treating or preventing cancer in a subject in need of treatment or prevention of cancer, optionally wherein the blood of the subject before said treatment or prevention contains at least 5 ng / ml of sCD28, and optionally wherein said cancer is selected from melanoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, renal cancer, gastric cancer and colorectal cancer, the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14.

16. An antibody or antigen-binding fragment thereof for use in improving an immune therapy based on PD-1 or PD-L1 in a subject in need of an immune therapy based on PD-1 or PD-L1, optionally wherein the blood of the subject before said immune therapy contains at least 5 ng / ml of sCD28, and wherein the subject has cancer or the subject does not respond or has a low response to said immune therapy, the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14.

17. The antibody or antigen-binding fragment thereof according to claim 15 or 16, wherein said use does not degrade mCD28 or reduce mCD28-mediated immune cell activation.

18. A method for generating an agent that binds to membrane CD28 (mCD28) and inhibits proteolytic cleavage of said mCD28, said method comprising: a. obtaining an agent that binds to the CD28 stalk region consisting of SEQ ID NO: 10, testing the ability of said agent to block cleavage of mCD28 by a protease, and selecting at least one agent that blocks cleavage of mCD28 by said protease, and optionally assaying mCD28 downstream signaling in the presence of said obtained agent and selecting at least one agent that does not substantially activate or antagonize mCD28 signaling; or b. culturing a host cell containing one or more vectors containing a nucleic acid sequence encoding an agent, said nucleic acid sequence comprising: i. obtaining an agent that binds to the CD28 stalk region consisting of SEQ ID NO: 10; ii. testing the ability of said agent to block cleavage of mCD28 by a protease; iii. selecting at least one agent that blocks cleavage of mCD28 by said protease; and optionally, iv. Assaying mCD28 downstream signaling in the presence of the obtained agent, and selecting at least one agent that neither substantially activates nor substantially antagonizes mCD28 signaling which is of the agent selected by comprising a method for generating an agent that binds to membrane CD28 (mCD28) and inhibits proteolytic cleavage of said mCD28 thereby.

19. obtaining said agent is a. immunizing an organism with the CD28 stalk domain and collecting antibodies from the immunized organism, optionally, the CD28 stalk domain being a dimer or monomer, the organism being selected from rabbit, mouse, rat, shark, camelid, chicken, and goat, or collecting said antibodies comprising extracting B cells from the spleen of the immunized organism, fusing the extracted B cells with myeloma cells to generate hybridomas, and collecting antibodies from the hybridomas, immunizing an organism with the CD28 stalk domain and collecting antibodies from the immunized organism; b. screening a library of agents for binding to the CD28 stalk domain and selecting an agent that binds, optionally, (i) selecting said binding agent comprising sequencing said selected agent and generating a recombinant form of said agent from said sequence, or (ii) said library being a phage library or both (i) and (ii), screening a library of agents for binding to the CD28 stalk domain and selecting an agent that binds comprising at least one of the method according to claim 18.

20. A method for generating an agent that binds to soluble CD28 (sCD28), does not bind to membrane CD28 (mCD28), and is neither a CD28 agonist nor an antagonist, said method comprising a. obtaining an agent that binds to the CD28 extracellular domain or a fragment thereof, assaying mCD28 downstream signaling in the presence of the obtained agent, and selecting at least one agent that neither substantially activates nor substantially antagonizes mCD28 signaling b. Testing the binding of the obtained agent to mCD28, selecting at least one agent that does not bind to mCD28, and optionally testing the binding of the obtained agent to sCD28 from cancer patients and selecting at least one agent that binds to said sCD28 from cancer patients; or c. Culturing a host cell containing one or more vectors comprising a nucleic acid sequence encoding an agent, wherein the nucleic acid sequence i. Obtaining an agent that binds to the CD28 extracellular domain or a fragment thereof; ii. Assaying mCD28 downstream signaling in the presence of the obtained agent; iii. Selecting at least one agent that neither substantially activates nor substantially antagonizes mCD28 signaling, and iv. Testing the binding of the obtained agent to mCD28, selecting at least one agent that does not bind to mCD28, and optionally testing the binding of the obtained agent to sCD28 from cancer patients and selecting at least one agent that binds to said sCD28 from cancer patients selected by the method, comprising thereby generating an agent that binds to sCD28, does not bind to mCD28, and is neither a CD28 agonist nor an antagonist.

21. Obtaining the agent is a. Immunizing an organism with the CD28 extracellular domain or a fragment thereof and collecting antibodies from the immunized organism, optionally wherein the CD28 extracellular domain or a fragment thereof is a dimer or a monomer, and the organism is selected from rabbit, mouse, rat, shark, camelid, chicken, and goat, or collecting the antibodies comprises extracting B cells from the spleen of the immunized organism, fusing the extracted B cells with myeloma cells to generate hybridomas, and collecting antibodies from the hybridomas, immunizing an organism with the CD28 extracellular domain or a fragment thereof and collecting antibodies from the immunized organism; b. Screening a library of agents for binding to the CD28 extracellular domain or a fragment thereof and selecting an agent that binds, optionally, (i) selecting the binding agent comprises sequencing the selected agent and generating a recombinant form of the agent from the sequence, or (ii) the library is a phage library or both (i) and (ii), screening a library of agents for binding to the CD28 extracellular domain or a fragment thereof and selecting an agent that binds The method according to claim 20, comprising at least one of the above.

22. At least one of the antibodies or antigen-binding fragments thereof according to any one of claims 1 to 14, and a. An anti-PD-1 and / or PD-L1 immune checkpoint inhibitor; b. A label indicating that the agent of the present invention is for use in combination with an anti-PD-1 and / or PD-L1 immune checkpoint inhibitor; and c. A secondary detection molecule for detecting at least one of the antibodies or antigen-binding fragments thereof according to any one of claims 1 to 15, a kit comprising at least one of the above.

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