DESCRIPTION

TECHNICAL HELD

This application relates to fusion proteins comprising extracellular domain of PD-1 (programmed cell death-1 protein) and/or fusion proteins comprising extracellular domain of PD-L1 (programmed cell death-ligand 1 protein) (CD274 or B7-H1). Portions of the extracellular domains are expressed in specific configurations and purified as protein and used in immunoassays to monitor the circulating levels of biotherapeutic antibodies to these proteins. Also described herein are methods for detecting and monitoring a therapeutic antibody in a biological sample obtained from a patient, wherein a patient has undergone at least one dose of immunotherapy.

BACKGROUND

Programmed cell death 1 (PD-1) receptor, one of the most studied immune checkpoint targets, is expressed on the surface of activated immune cell types, including T cells, B cells, natural killer (NK)/natural killer T (NKT) cells and others'. Multiple therapeutic monoclonal antibodies (mAbs) targeting PD-1 and PD-L1 are on the market and more than a dozen traditional and bispecific mAbs are being reviewed at the FDA and EMA.

PD-1 is a negative regulator of T-cell activity and the interaction of PD-1 with one of its ligands, PD-L1 , at the tumor surface produces a checkpoint blockade decreasing the ability of activated T cells to produce an effective immune response. The expression of high levels of PD-L1 on tumor cell surfaces, permits escape from the anti-tumor responses by suppressing T cell functions, including cytotoxic activity. Therapeutic antibodies that target either PD-1 or PD-L1 will block the ligand-receptor interaction, and restore immune function to the tumor microenvironmenL The use of these mAbs has yielded exciting clinical responses across many cancer types and a growing number of these mAbs have now entered clinical development

Due to the novelty of targeting the immunological checkpoint blockade activated by cancer cells, optimal dose and schedule for administering these mAbs and the tools necessary to determine these parameters have not been fully developed. While current anti-PD-1 mAb therapeutic regimens have shown promising results, it will be necessary to define more accurate and personalized, dosing regimens to increase efficacy and decrease cost Steady state antibody levels can vary up to 10-fold for the same antibody regimen, and even more so when sub-therapeutic dosing is used ^2' *. These studies could optimize treatment while decreasing cost burden and adverse event incidence. A large number of immuno- modulatory mAbs are under review by the FDA and EU, many on a fast track basis, further highlighting the need for tests which measure circulating levels of PD-1 and PD-L1 targeted mAbs. The immunoassays under development will provide effective guidance of treatment regimens, particularly given (1) the gross variation observed in steady state mAb levels both within and across disease indications and (2) the varying progression states that are observed in patients under these therapies ^1,3,5 .

Measuring anti-PD-1 and anti-PD-L1 levels has potential importance not only in determining optimal dosing for different tumors or molecular pathologies, but also to gauge whether circulating levels may explain or predict adverse events known to occur following remission. There is an unmet need because no monitoring programs currently exist and more data is required to evaluate and determine dosing schedules and obviate unnecessary immunotoxicity and cost. Nivolumab is a humanized lgG4 anti-PD-1 mAb. Nivolumab works as a checkpoint inhibitor, blocking a negative regulator that would have prevented activated T cells from attacking the cancer, thus allowing the immune system to clear the cancer. It was discovered at Medarex, developed by Medarex and Ono Pharmaceutical, and brought to market by Bristol-Myers Squibb (which acquired Medarex in 2009) and Ono.

Nivolumab (Opdivo®), is a biotherapeutic mAb used to treat cancer and first approved by the Japanese authorities in 2014 ^e and by the FDA also in 2014 ⁷ . It is used as a front-line treatment for inoperable or metastatic skin melanoma It is used differentially in combination with ipilimumab (mAb against CTLA-4, cytotoxic T-lymphocyte associated protein-4) if the cancer does or does not have a mutation in BRAF. It is considered to be used as a second-line treatment for squamous non-small cell lung cancer (NSCLC) ⁸ , and as a second-line treatment for renal cell carcinoma ⁹ and in May 2016 was approved for recalcitrant classical Hodgkin Lymphoma. ⁹ It is being tested for many other cancers and likely will have greatly expanded uses. Merck produces a similar mAb therapy pembrolizumab (Keytruda), also an lgG4 class mAb against PD-1 , which was also approved in 2014. As of 2017, pembrolizumab is used via intravenous infusion to treat inoperable or metastatic melanoma, metastatic NSCLC in certain situations, as a second-line treatment for head and neck squamous cell carcinoma, refractory classical Hodgkin's lymphoma. For NSCLC, Pembrolizumab is a first line treatment if the cancer overexpresses PD-L1 and the cancer has no mutations in EGFR or in ALK; assessment of PD-L1 must be conducted with a validated and approved companion diagnostic. Moreover, Pembrolizumab is the first drug approved by the FDA based on the presence of a biomarker (PD-1) instead of the targeted tissue. ¹⁰ Many other anti-PD-1 and antj-PD-L1 biotherapeutic antibodies are in the clinic now and include alternative formats such as bispecifics, DARTs (Dual Anti-CTLA-4 & Anti-PD-1 blockade in Rare Tumors) and BiTes (Bispecific T cell engagers).

Conversely, upregulation of PD-L1 allows cancers to evade the host immune system and high tumor expression of PD-L1 is associated with increased tumor aggressiveness and a 4.5-fold increased risk of death. ¹¹ In addition to the PD-L1 inhibitors already approved for treatment, many PD-L1 are in development as immuno-oncology therapies and are showing promising results in clinical trials including Atezolizumab and Avelumab.

These drugs increase the efficacy of T cells which are able to more aggressively attack and kill cancer cells. For Nivolumab, in some cases there are severe side effects that include immune-related inflammation of the lungs, colon, liver, kidneys, and thyroid; and effects on skin, central nervous system, heart, and digestive system. ¹² For Pembrolizumab, patients have developed severe infusion-related reactions and adverse effects including lung inflammation (some fatal) and inflammation of endocrine organs that caused inflammation (pituitary gland, thyroid (causing both hypothyroidism and hyperthyroidism in different patients), and pancreatitis that caused Type 1 diabetes and diabetic ketoacidosis) resulting in some patients going on lifelong hormone therapy (e.g. insulin therapy or thyroid hormones). Patients have also developed colon, liver, and kidney inflammation due to the mAb drug.' ³ Many other symptoms and malaise are reported for both. Patients who show these side effects are taken off the mAb drugs, then put back on the drug if / when the side effects subside. It is possible that the doses being used could be titered or greatly reduced if the level in a patient were monitored. However, there are currently no monitoring assays available for testing the circulating mAb drug levels and no antigens for these tests have been qualified. SUMMARY

The present application provides fusion proteins related to programmed cell death 1 (PD-1) and programmed cell death-ligand (PD-L1) proteins. The fusion protein can be a recombinant protein comprising either a modification of the natural protein and/or a fusion protein comprised of another domain with distinct properties to improve function in a diagnostic assay.

One aspect described herein provides a recombinant protein comprising a PD-1 protein fused to an oligomerization domain. Another aspect described herein provides a fusion protein comprising a PD-1 protein fused to oligomerization domains. Another aspect described herein provides a recombinant protein comprising a PD-L1 protein fused to an oligomerization domain. Another aspect described herein provides a fusion protein comprising a PD-L1 protein fused to oligomerization domains. Yet another aspect described herein provides a fusion protein comprising PD-1 and PD-L1 protein fused to an oligomerization domain. In some embodiments, the fusion protein is a recombinant protein. The PD-1 and/or PD-L1 proteins may be from multiple species or from a single species. In one aspect, the PD-1 and/or PD-L1 protein(s) is a human PD-1 and/or human PD-L1 protein(s). In another aspect, the fusion protein comprises the extracellular domain of the PD-1 and/or PD-L1 protein or a fragment thereof. In one embodiment, the fusion protein comprises an immunoglobulin-like domain of the extracellular domain of the PD-1. In another embodiment, the fusion protein comprises an immunoglobulin-like domain of the extracellular domain of the PD-L1.

The PD-1 and/or PD-L1 extracellular domain(s) or a fragment thereof may comprise various modified proteins or peptides. The modification may be substitution, deletion or addition of at least one amino acid in wild-type PD-1 and/or PD-L1, as long as it does not change the function of PD-1 and PD-L1. Such various proteins or peptides may have a sequence homology of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to the wild-type protein. The fusion proteins described herein comprise an extracellular domain of PD-1 or PD-L1 proteins, or a fragment thereof, and an oligomerization domain.

In some aspects, the fusion protein described herein comprises an extracellular domain of PD-1 protein, or a fragment thereof, and an oligomerization domain, wherein the oligomerization domain is selected from the group consisting of a murine lgG1 Fc domain, a murine lgG2A Fc domain, a GCN4 trimer domain, a clathrin trimer domain, and a p53 tetramer domain, or a fragment thereof.

In other aspects, the fusion protein described herein comprises an extracellular domain of PD-L1 protein, or a fragment thereof, and an oligomerization domain, wherein the oligomerization domain is selected from the group consisting of a murine lgG1 Fc domain, a murine lgG2A Fc domain, a GCN4 trimer domain, a clathrin trimer domain, and a p53 tetramer domain, or a fragment thereof.

The oligomerization domain described herein may comprise a domain selected from the group consisting of Clathrin, GCN4 trimerization domains, the p53 tetramerization domain, and pentameric domains. In some aspects, the oligomerization domain may comprise an Fc domain, a fragment of an Fc domain, and a variant of an Fc domain. In one embodiment, the PD-1 and/or PD-L1 protein is fused to the C-terminus of said Fc domain, or fragment thereof. In another embodiment, the PD-1 and/or PD-L1 protein is fused to the N-terminus of said Fc domain, or fragment thereof. In one embodiment, the Fc domain may be of human or mouse immunoglobulin. In another embodiment, the oligomerization domain may comprise an Fc fragment of mouse lgG1 or mouse lgG2A, or fragments thereof.

In some aspects, the oligomerization domain of the fusion protein described herein is a murine lgG1 Fc domain or a murine lgG2A Fc domain. In some aspects, the murine lgG1 Fc domain or the murine lgG2A Fc domain comprises a hinge region.

Fusion proteins that comprise an extracellular domain of PD-1 protein, or a fragment thereof, and an oligomerization domain, as described herein, may comprise a murine lgG1 Fc domain or a murine lgG2A Fc domain which comprises a hinge region.

Fusion proteins that comprise an extracellular domain of PD-L1 protein, or a fragment thereof, and an oligomerization domain, as described herein, may comprise a murine lgG1 Fc domain or a murine lgG2A Fc domain which comprises a hinge region. The fusion protein described herein can be part of a higher order structure, such as a protein or multjmeric complex. This can be either through intentional design of the protein with multimerization domains or inherently, via unexpected properties of the recombinant protein. In some aspects, the PD-1 and/or PD-L1 protein, or the fragment thereof, is fused to the oligomerization domain, via one or more peptide linkers. In some aspects, the extracellular domain of PD-1 protein, or the fragment thereof, is fused to the oligomerization domain, via one or more peptide linkers. In some aspects, the extracellular domain of PD-L1 protein, or the fragment thereof, is fused to the oligomerization domain, via one or more peptide linkers. In some aspects, the PD-1 and/or PD-L1 protein is fused to the Fc domain, or fragment thereof, via one or more peptide linkers. In some aspects, in the fusion proteins described herein, the extracellular domain of PD-1 protein, or the fragment thereof, is fused to the oligomerization domain via one or more flex linkers comprising amino acids Glycine and Serine or via one or more flex pro linkers comprising amino acids Glycine, Proline, and Serine. In some aspects, said one or more peptide linkers comprise the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5. In some aspects, in the fusion proteins described herein, the extracellular domain of PD-L1 protein, or the fragment thereof, is fused to the oligomerization domain via one or more flex linkers comprising amino acids Glycine and Serine or via one or more flex pro linkers comprising amino acids Glycine, Proline, and Serine. In some aspects, said one or more peptide linkers comprise the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5.

In some embodiments, the fusion proteins may comprise the amino acid sequence set forth in SEQ ID NOs: 14-27 or SEQ ID NOs: 29-30.

Other aspects of the present invention include fusion proteins as described herein, further comprising a signal sequence, wherein the signal sequence is fused at the N-terminus of the extracellular domain of PD-1 protein or the fragment thereof. Other aspects of the present invention include fusion proteins as described herein, further comprising a signal sequence, wherein the signal sequence is fused at the N- terminus of the extracellular domain of PD-L1 protein or the fragment thereof. In some aspects the signal sequence comprises the amino acid sequence set forth in SEQ ID NO: 3. In other aspects, the amino acid sequence of the signal sequence shares at least 70% homology with the amino acid sequence set forth in SEQ ID NO: 3.

In some aspects of the present invention, the fusion protein described herein comprises an extracellular domain of PD-1 which amino acid sequence shares at least 70% homology with the amino acid sequence consisting of residues 21 to 170, 21 to 145, 33 to 170, 33 to 145, 35 to 170 or 35 to 145 of SEQ ID NO:1. In some aspects, the fusion protein comprises an extracellular domain of PD-1 which amino acid sequence shares at least 70% homology with the amino acid sequence consisting of residues 21 to 170 of SEQ ID NO:1. In some aspects, the fusion protein comprises an extracellular domain of PD- 1 which amino acid sequence consists of residues 21 to 170 of SEQ ID NO:1.

In other aspects of the present invention, the fusion protein described herein comprises an extracellular domain of PD-L1 which amino acid sequence shares at least 70% homology with the amino acid sequence consisting of residues 18 to 239, 18 to 238, 18 to 225, 18 to 134, 18 to 127, 19 to 239, 19 to 238, 19 to 225, 19 to 134, 19 to 127, 133 to 225, 133 to 238, or 133 to 239 of SEQ ID NO: 2. In some aspects, the fusion protein comprises an extracellular domain of PD-L1 which amino acid sequence shares at least 70% homology with the amino acid sequence consisting of residues 18 to 239 of SEQ ID NO:2. In some aspects, the fusion protein comprises an extracellular domain of PD-L1 which amino acid sequence consists of residues 18 to 239 of SEQ ID NO:2.

In yet another aspect, the fusion protein as described herein comprises an extracellular domain of PD-1 which amino acid sequence comprises a sequence with at least 70% homology with the amino acid sequence set forth in SEQ ID NO: 12. In yet another aspect, the fusion protein as described herein comprises an extracellular domain of PD-L1 which amino acid sequence comprises a sequence with at least 70% homology with the amino acid sequence set forth in SEQ ID NO: 22.

The fusion protein according to the present invention may comprise an extracellular domain of PD-1, or fragment thereof, and an oligomerization domain which amino acid sequence is selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , and SEQ ID NO:28.

The fusion protein according to the present invention may comprise an extracellular domain of PD-L1, or fragment thereof, and an oligomerization domain which amino acid sequence is selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , and SEQ ID NO:28.

In some aspects, the amino acid sequence of the fusion protein described herein is selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 29. In some aspects, the amino acid sequence of the fusion protein described herein is selected from the group consisting of the amino acid sequence that shares at least 70% homology with the amino acid sequence set forth in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 29.

In some aspects, the amino acid sequence of the fusion protein described herein is selected from the group consisting of SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 30. In some aspects, the amino acid sequence of the fusion protein described herein is selected from the group consisting of the amino acid sequence that shares at least 70% homology with the amino acid sequence set forth in SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 30. Some aspects of the present invention relate to fusion proteins as described herein wherein the PD-1 is recombinant PD-1. Yet other aspects of the present invention relate to fusion proteins as described herein wherein the PD-1 is recombinant human PD-1.

Some aspects of the present invention relate to fusion proteins as described herein wherein the PD-L1 is recombinant PD-L1. Yet other aspects of the present invention relate to fusion proteins as described herein wherein the PD-L1 is recombinant human PD-L1.

One aspect described herein provides a nucleic acid encoding a recombinant fusion protein, a vector comprising the nucleic acid, and a host cell comprising the vector. In one embodiment, the vector encodes a nucleic acid molecule linked to a signal peptide sequence.

Another aspect described herein provides a method of making a fusion protein comprising the extracellular domain of PD-1 and/or PD-L1 protein, said method comprising: (a) transfecting a host cell with a nucleic acid molecule encoding the fusion protein, wherein the nucleic acid molecule comprises a nucleotide sequence encoding a signal peptide, fused to either i) a nucleotide sequence encoding an Fc domain of a human or mouse IgG linked to a nucleotide sequence encoding the extracellular domain of PD-1 and/or PD-L1 peptide, at the N-terminus of said extracellular domain of PD-1 and/or PD-L1 peptide, or ii) a nucleotide sequence encoding the PD-1 and/or PD-L1 peptide linked to a nucleotide sequence encoding an Fc domain of a human or mouse or other mammalian IgG, at the N-terminus of said Fc domain, and (b) making the fusion protein by expressing the nucleic acid molecule of (a) in the host cell. In one embodiment, the extracellular domain is fused to the Fc domain via a linker. The linker may be SEQ ID NO: 4 or SEQ ID NO: 5. Another aspect describe herein provides a method of making a fusion protein comprising the extracellular domain of PD-1 and/or PD-L1 protein, said method comprising: (a) transfecting a host cell with a nucleic acid molecule encoding the fusion protein as described herein, and (b) making the fusion protein by expressing the nucleic acid molecule of (a) in the host cell.

For recombinant production of the fusion protein, a wide variety of expression vectors can be constructed based on common molecular cloning protocols. The vector components generally include, but are not limited to, one or more of the following: a signal sequence for the secretion of expressed proteins, one or more marker genes including the selection marker gene for the stable cell line screening in eukaryote cells, an origin of replication, an enhancer element, a promoter, a transcription termination sequence, a poly A signal, an insulator, etc.

Also provided herein is a composition comprising a fusion protein comprising PD-1 and/or PD-L1 , or a fragment thereof, and an digomerization domain. Further provided herein is a method of making a composition comprising a fusion protein comprising PD-1 and/or PD-L1 or a fragment thereof and an oligomerization domain as described herein. A kit comprising the fusion protein or the composition is also provided.

In addition, provided herein are methods for antibody biomonitoring using the fusion proteins as antigens. The fusion proteins described herein can serve as antigen(s) for therapeutic antibody biomonitoring. The antibody includes anti-PD-1 (such as Nivolumab and Pembrolizumab) and/or anti-PD-L1 antibodies (Atezolizumab and Avelumab).

One aspect described herein provides a method for diagnosing, monitoring or staging diseases mediated by PD-1 and/or PD-L1 comprising contacting a sample from a patient, having undergone at least one dose of immunotherapy, with a fusion protein disclosed herein.

One aspect described herein provides a method for determining the efficacy of therapeutic anti-PD-1 and/or PD-L1 antibodies comprising contacting a sample from a patient, having undergone at least one dose of immunotherapy, with a fusion protein disclosed herein. Another aspect described herein includes a method of providing an immunotherapy to a patient comprising administering to the patient a therapeutic antibody and detecting the presence of anti-PD-1 and/or anti-PD-L1 therapeutic antibodies. It is envisioned that these methods can be used to monitor the efficacy of antibody-based therapy. Another aspect described herein includes methods for detecting and/or monitoring diseases mediated by PD-1 and/or PD-L1 by measuring anti-PD-1 and/or anti-PD-U therapeutic antibodies. In some aspects the method comprises contacting a sample from a patient with a fusion protein described herein. In some aspects, , the patient has undergone a course of immunotherapy with a therapeutic antibody.

In some aspects the present invention relates to a method of determining the amount of circulating levels of a biotherapeutjc antibody selected from the group consisting of nivolumab, pembrolizumab and other anti-PD-1 therapies, comprising (i) obtaining a sample from a patient undergoing the antibody treatment, and (ii) contacting the sample with the fusion protein described herein.

In some aspects the present invention relates to a method of determining the amount of (free) circulating levels of a biotherapeutic antibody selected from the group consisting of atezolizumab, avelumab, and other anti PD-L1 therapies, comprising (i) obtaining a sample from a patient undergoing the antibody treatment, and (ii) contacting the sample with the fusion protein described herein.

The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

The foregoing has outlined rather broadly the features and technical advantages of the embodiments disclosed herein in order that the detailed description that follows may be better understood. Additional features and advantages will be described hereinafter which form the subject of the claims. It should be appreciated by those skilled in the art that the conception and specific embodiment disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of various embodiments disclosed herein. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be characteristic of various embodiments disclosed herein, both as to their organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the disclosed embodiments. BRIEF DESCRIPTION OF DRAWINGS

Figure 1 - Represents SDS-PAGE chromatography of various fusion proteins post expression and purification with a reducing 4-20% Tris-glycine TGX gel. Figure 2 - Represents SDS-PAGE chromatography of various fusion proteins post expression and purification with a non-reducing 4-20% Tris-glycine TGX gel Figure 3A - Shows specific reactivity of a recombinant PD-1 fusion protein against anti-PD-1 antibody in a serial dilution series (dilutions from 0.5ug/ml to 0.04u.g/ml).

Figure 3B - Shows specific reactivity of recombinant PD-L1 fusion proteins against anti-PD-L1 antibody in a dilution series as mentioned above. The figure also shows a comparison of the reactivity of PD-L1 -Fc monomer and PD-L1-p53 tetramer.

Figure 4A - Shows a comparison of various PD-1 fusion proteins for quantification of anti-PD-1 antibodies (pembrolizumab) in human plasma.

Figure 4B - Shows the signal to cut off noise of various PD-1 fusion proteins for quantification of anti-PD- 1 antibodies (pembrolizumab) in human plasma

Figure 5 - Shows specific reactivity of various PD-L1 fusion proteins against anti-PD-L1 antibodies.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is the amino acid sequence of PD-1 (UniProt: Q15116). SEQ ID NO: 2 is the amino acid sequence of PD-L1 (UniProt: Q9NZQ7) SEQ ID NO: 3 is the amino acid sequence of Human IL2 signal peptide. SEQ ID NO: 4 is the amino acid sequence of flexible Gly-Ser linker.

SEQ ID NO: 5 is the amino acid sequence of flexible Gly-Pro-Ser linker (Rex Pro Linker). SEQ ID NO: 6 is the amino acid sequence of Murine lgG1 containing CH2 and CH3 domains only. SEQ ID NO: 7 is the amino acid sequence of Murine lgG1 containing Fc region. SEQ ID NO: 8 is the amino acid sequence of Murine lgG2A containing Fc region. SEQ ID NO: 9 is the amino acid sequence of GCN4 trimer domain.

SEQ ID NO: 10 is the amino acid sequence of Clathrin trimer domain. SEQ ID NO: 11 is the amino acid sequence of p53 tetramer domain. SEQ ID NO: 12 is the amino acid sequence of PD-1 fragment. SEQ ID NO: 13 is the amino acid sequence of PD-L1 fragment. SEQ ID NO: 14 is the amino acid sequence of a PD-1 fusion protein (HulL2SS-PD-1(GGGGS)2-Murine lgG1 (CH2, CH3 ONLY) 362 AA Flex Linker) according to one embodiment of the invention.

SEQ ID NO: 15 is the amino acid sequence of a PD-1 fusion protein (HulL2SS-PD-1 (GGGPS)2- Murine lgG1 (CH2, CH3 ONLY) 362 AA Flex Pro Linker) according to another embodiment of the invention.

SEQ ID NO: 16 is the amino acid sequence of a PD-1 fusion protein (HulL2SS-PD-1 - murine lgG1 Fc 365 AA No hinge) according to yet another embodiment of the invention.

SEQ ID NO: 17 is the amino acid sequence of a PD-1 fusion protein (HulL2SS-PD-1 -murine lgG2A Fc 370 AA No hinge) according to yet another embodiment of the invention.

SEQ ID NO: 18 is the amino acid sequence of a PD-1 fusion protein (HulL2SS-PD-1-GCN4 trimer domain) according to yet another embodiment of the invention.

SEQ ID NO: 19 is the amino acid sequence of a PD-1 fusion protein (HulL2SS-PD-1-Clathrin trimer domain) according to yet another embodiment of the invention.

SEQ ID NO: 20 is the amino acid sequence of a PD-1 fusion protein (HulL2SS-PD-1-p53 tetramer domain) according to yet another embodiment of the invention.

SEQ ID NO: 21 is the amino acid sequence of a PD-L1 fusion protein (HulL2SS-PD-L1(GGGGS)2- Murine lgG1 (CH2, CH3 ONLY) 361 AA Flex linker) according to one embodiment of the invention.

SEQ ID NO: 22 is the amino acid sequence of a PD-L1 fusion protein (HulL2SS-PD-L1(GGGPS)2- Murine lgG1 (CH2, CH3 ONLY) 361 AA flex pro linker) according to another embodiment of the invention.

SEQ ID NO: 23 is the amino acid sequence of a PD-L1 fusion protein (HulL2SS-PD-L1 -murine lgG1 Fc 364 AA no hinge) according to yet another embodiment of the invention.

SEQ ID NO: 24 is the amino acid sequence of a PD-L1 fusion protein (HulL2SS-PD-L1 -murine lgG2A Fc 370 AA No hinge) according to yet another embodiment of the invention.

SEQ ID NO: 25 is the amino acid sequence of a PD-L1 fusion protein (HulL2SS-PD-L1-GCN4 trimer domain) according to yet another embodiment of the invention.

SEQ ID NO: 26 is the amino acid sequence of a PD-L1 fusion protein (HulL2SS-PD-L1-Clathrin trimer domain) according to yet another embodiment of the invention.

SEQ ID NO: 27 is the amino acid sequence of a PD-L1 fusion protein (HulL2SS-PD-L1-p53 tetramer domain) according to yet another embodiment of the invention.

SEQ ID NO: 28 is the amino acid sequence of Murine lgG2A containing CH2 and CH3 domains only. SEQ ID NO: 29 is the amino acid sequence of a PD-1 fusion protein (HulL2SS-PD-1(GGGGS)2-p53- Histag) according to one embodiment of the invention. SEQ ID NO: 30 is the amino acid sequence of a PD-L1 fusion protein (HulL2SS-PD-L1(GGGGS)2-p53- Histag) according to one embodiment of the invention.

SEQ ID NO: 31 is the amino acid sequence of a polyhistidine tag according to one embodiment of the invention.

DETAILED DESCRIPTION

The following description is merely intended to illustrate various embodiments of the present disclosure. As such, the specific modifications discussed are not intended to be limiting. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the spirit or scope of the subject matters presented herein, and it is understood that such equivalent embodiments are to be included herein.

As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural references unless the content clearly dictates otherwise.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

Each embodiment in this specification is to be applied mutatis mutandis to every other embodiment unless expressly stated otherwise. The following terms, unless otherwise indicated, shall be understood to have the following meanings:

As used herein, the term "recombinant" refers to a biomolecule, e.g., a gene or protein, that (1) has been removed from its naturally occurring environment, (2) is not associated with all or a portion of a polynucleotide in which the gene is found in nature, (3) is operatively linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature. The term "recombinant" can be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs that are biologically synthesized by heterologous systems, as well as proteins and/or mRNAs encoded by such nucleic acids. As used herein, the term "nucleic acid" refers to any materials comprised of DNA or RNA. Nucleic acids can be made synthetically or by living cells. As used herein, the term "polynucleotide" refers to a polymeric chain of nucleotides. The term includes DNA molecules (e.g., cDNA or genomic or synthetic DMA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA or RNA containing non-natural nucleotide analogs, non-native inter- nucleoside bonds, or both. The nucleic acid can be in any topological conformation. For instance, the nucleic acid can be single-stranded, double-stranded, triple-stranded, quadruplexed, partially double- stranded, branched, hair-pinned, circular, or in a padlocked conformation.

As used herein, the term "protein" refers to large biological molecules, or macromolecules, consisting of one or more chains of amino acid residues. Many proteins are enzymes that catalyze biochemical reactions and are vital to metabolism. Proteins also have structural or mechanical functions, such as actin and myosin in muscle and the proteins in the cytoskeleton, which form a system of scaffolding that maintains cell shape. Other proteins are important in cell signaling, immune responses, cell adhesion, and the cell cycle. However, proteins may be completely artificial or recombinant, i.e., not existing naturally in a biological system.

As used herein, the term "polypeptide" refers to both naturally-occurring and non-naturally-occurring proteins, and fragments, mutants, derivatives and analogs thereof. A polypeptide may be monomelic or polymeric. A polypeptide may comprise a number of different domains each of which has one or more distinct activities. A polypeptide may be a fusion protein, as described herein.

As used herein, the term "fusion protein" refers to proteins comprising two or more amino acid sequences that do not co-exist in naturally-occurring proteins. A fusion protein may comprise two or more amino acid sequences from the same or from different organisms. The two or more amino acid sequences of a fusion protein are typically in frame without stop codons between them and are typically translated from mRNA as part of the fusion protein.

As used herein, the term "antigen" refers to a biomolecule that binds specifically to the respective antibody. An antibody from the diverse repertoire binds a specific antigenic structure by means of its variable region interaction (CDR loops), an analogy being the fit between a lock and a key.

The term "variant," when used in the context of a polynucleotide sequence, may encompass a polynucleotide sequence related to a wild type gene. This definition may also include, for example, "allelic", "splice," "species," or "polymorphic" variants. A splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during MRNA processing. The corresponding polypeptide may possess additional functional domains or an absence of domains. Species variants are polynucleotide sequences that vary from one species to another. Of particular utility in the invention are variants of wild type target gene products. Variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. Any given natural or recombinant gene may have none, one, or many allelic forms. Common mutational changes that give rise to variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence. The terms "antibody" or "immunoglobulin", as used herein, have the same meaning, and will be used equally in the present invention. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immune specifically binds an antigen. As such, the term antibody encompasses not only whole antibody molecules, but also antibody fragments or derivatives. Antibody fragments include but are not limited to Fc, Fv, Fab, F(ab') ₂ , Fab', dsFv, scFv, sc(Fv) ₂ and diabodies.

In natural antibodies, two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (λ) and kappa (κ). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. The light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1, CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). -Occasionally, residues from non-hypervariable or framework regions (FR) influence the overall domain structure and hence the combining site. Complementarity Determining Regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1 , L-CDR2, L-CDR3 and H-CDR1 , H-CDR2, H-CDR3, respectively. An antigen-binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. Framework Regions (FRs) refer to amino acid sequences interposed between CDRs.

Many approved therapeutic antibodies have used the G1m17.1 :Km3 heavy and light chain constant region allotypes (Jeffris and Lefranc 2009). Others use alternative G1m3:Km3 allotypes. The role of allotype in rejection of therapeutic antibodies was not apparent at the outset of mAb therapy as differences in allotypy were not known to be as common as they are today. There is a clear link between the allotype of therapeutic mAbs and clearance. This is due to the interaction differences with the FcRn neonatal Fc receptor which function to keep some of the mAb sequestered within cellular compartments to be recycled back into the blood stream and not cleared rapidly (Ternant et al 2016) . These difference in allotypy and the effects on cellular immune stimulation are becoming more known today (Webster et al 2016) . While unlikely to generate immunogenicity they have a clear effect on half-life. Thus tests to measure the amount of circulating therapeutic antibody can reveal levels of drug and potential needs for dose escalation. Alternatively, some patients may require less drug to maintain an effective level in particular if the level of anti-PD-1 on the T cell surface maintains a high level. These antigens can be used to measure binding by any type of antibody (lgG1, lgG4, DARTs, BiTes etc) if used in conjunction with specific reagents or in competition assays familiar to those schooled in the art of Immunoassay.

Next generation combination therapy using multiple immune checkpoint inhibitor mAbs and other drugs also require effective means of monitoring levels. These types of combination treatments are not only more effective but are becoming common (Dempke et al 2017). Assays described herein will also be important to measure the anti-PD-1 or antj-PD-L1 components in these cases.

As used herein, "site-directed mutagenesis" refers to a process in which a mutation is created at a defined site in a DNA molecule. The defined site refers to sites chosen as based on the affinity of interaction. The DNA molecule for mutagenesis usually is a circular molecule known as a plasmid. In general, site-directed mutagenesis requires that the wild-type gene sequence be known. This technique is also known as "site-specific mutagenesis" or "oligonucleotide-directed mutagenesis". Consequently, "site-directed mutation" means mutations created at a defined site in a DNA molecule by technique of site-directed mutagenesis. In one embodiment, a mutant DNA sequence generated using site directed mutagenesis have more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, or more than 98% homology with the wild-type sequence. Alternatively, mutant DNA may be generated in vivo using any known mutagenic procedure (e.g., radiation, nitrosoguanidine, etc.). The DNA construct sequences may be wild-type, mutant or modified. In addition, the sequences may be homologous or heterologous.

The terms "wild-type sequence" or "wild-type gene" are used interchangeably herein, to refer to a sequence that is native or naturally occurring in a host cell. In some embodiments, the wild-type sequence refers to a sequence of interest that is the starting point of a protein engineering project The wild-type sequence may encode either a homologous or heterologous protein. A homologous protein is one the host cell would produce without intervention. A heterologous protein is one that the host cell would not produce but for the intervention. The terms "modified sequence" and "modified genes" are used interchangeably herein to refer to a sequence that includes a deletion, insertion or interruption of naturally occurring nucleic acid sequence. In some preferred embodiments, the expression product of the modified sequence is a truncated protein (e.g., if the modification is a deletion or interruption of the sequence). In some particularly preferred embodiments, the truncated protein retains biological activity. In alternative embodiments, the expression product of the modified sequence is an elongated protein (e.g., modifications comprising an insertion into the nucleic acid sequence). In some embodiments, an insertion leads to a truncated protein (e.g., when the insertion results in the formation of a stop codon). Thus, an insertion may result in either a truncated protein or an elongated protein as an expression product. As used herein, the terms "mutant sequence" and "mutant gene" are used interchangeably and refer to a sequence that has an alteration in at least one codon occurring in a host cell's wild-type sequence. The expression product of the mutant sequence is a protein with an altered amino acid sequence relative to the wild-type. The expression product may have an altered functional capacity (e.g., enhanced binding affinity).

As used herein, the term linker" refers to a polypeptide comprising of 1-30 amino acids, preferably 3-6 amino acids. The amino acids of the linker may be selected from the group consisting of leucine (Leu, L), isoleucine (lie, I), alanine (Ala, A), glycine (Gly, G), valine (Val, V), proline (Pro, P), lysine (Lys, K), arginine (Arg, R), Serine (Ser, S), asparagine (Asn, N), and glutamine (Gin, Q), tryptophan (Trp, W), methionine (Met, M) aspartic acid (Asp, D), cysteine (Cys, C), glutamic acid (Glu, EE), histidine (His, H), phenylalanine (Phe, F), threonine (The, T), and tyrosine (Tyr, Y).

"Diagnostic" or "diagnosed" means identifying the presence or nature of a pathologic condition or a patient susceptible to a disease. Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives"). Diseased individuals not detected by the assay are "false negatives." Subjects who are not diseased and who test negative in the assay, are termed "true negatives." The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.

The terms "patient" or "individual" are used interchangeably herein, and refers to a mammalian subject to be treated, with human patients being preferred. In some cases, the methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; and primates.

The term "sample", as used herein, refers to any biological material obtained from a subject or patient In one aspect, a sample can comprise blood, peritoneal fluid, CSF, saliva or urine. In other aspects, a sample can comprise whole blood, blood plasma, blood serum, B cells enriched from blood samples, and cultured cells (e.g., B cells from a subject). A sample can also include a biopsy or tissue sample including neural tissue. In still other aspects, a sample can comprise whole cells and/or a lysate of the cells.

The term "treating" or "treatment", as used herein, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. A "therapeutically effective amount" is intended for a minimal amount of active agent which is necessary to impart therapeutic benefit to a subject. For example, a "therapeutically effective amount" to a mammal is such an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to a disorder.

The term "prevention", as used herein, refers to preventing the disease or condition from occurring in a subject which has not yet been diagnosed as having it. The terms "cancer", "hyperproliferative" and "neoplastic", as used herein, refer to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. Hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. The terms "cancer" or "neoplasms" include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.

One aspect described herein utilizes therapeutic antibodies to diagnose, monitor or stage diseases mediated by PD-1 and/or PD-L1 and efficacy of therapy. More particularly, methods of detecting and monitoring a therapeutic antibody, wherein a patient has undergone at least one course of immunotherapy, are disclosed. A variety of cancers or chronic infections can be monitored, staged or diagnosed according to the methods disclosed herein.

Examples of cancers include, but are not limited to, hematological malignancies such as B-cell lymphoid neoplasm, T-cell lymphoid neoplasm, non-Hodgkin lymphoma (NHL), B-NHL, T-NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), NK-cell lymphoid neoplasm and myeloid cell lineage neoplasm. Examples of non-hematological cancers include, but are not limited to, colon cancer, breast cancer, lung cancer, brain cancer, prostate cancer, head and neck cancer, pancreatic cancer, bladder cancer, colorectal cancer, bone cancer, cervical cancer, liver cancer, oral cancer, esophageal cancer, thyroid cancer, kidney cancer, stomach cancer, testicular cancer and skin cancer

Examples of chronic infections include, but are not limited to, viral, bacterial, parasitic or fungal infections such as chronic hepatitis, lung infections, lower respiratory tract infections, bronchitis, influenza, pneumoniae and sexually transmitted diseases. Examples of viral infections include, but are not limited to, hepatitis (HAV, HBV, HCV), herpes simplex (HSV), herpes zoster, HPV, influenza (Flu), AIDS and AIDS related complex, chickenpox (varicella), common cold, cytomegalovirus (CMV) infection, smallpox (variola), Colorado tick fever, dengue fever, ebola hemorrhagic fever, foot and mouth disease, lassa fever, measles, marburg hemorrhagic fever, infectious mononucleosis, mumps, norovirus, poliomyelitis, progressive multifocal leukencephalopathy (PML), rabies, rubella, SARS, viral encephalitis, viral gastroenteritis, viral meningitis, viral pneumonia, West Nile disease and yellow fever. Examples of bacterial infections include, but are not limited to, pneumonia, bacterial meningitis, cholera, diphtheria, tuberculosis, anthrax, botulism, brucellosis, campylobacteriosis, typhus, gonorrhea, listeriosis, lyme disease, rheumatic fever, pertussis (Whooping Cough), plague, salmonellosis, scarlet fever, shigellosis, syphilis, tetanus, trachoma, tularemia, typhoid fever, and urinary tract infections. Examples of parasitic infections include, but are not limited to, malaria, leishmaniasis, trypanosomiasis, chagas disease, cryptosporidiosis, fascioliasis, filariasis, amebic infections, giardiasis, pinworm infection, schistosomiasis, taeniasis, toxoplasmosis, trichinellosis, and trypanosomiasis. Examples of fungal infections include, but are not limited to, candidiasis, aspergillosis, coccidioidomycosis, cryptococcosis, histoplasmosis and tinea pedis.

The methods disclosed herein can be utilized to determine a dosage of a therapeutic anti-PD-1 and/or anti-PD-L1 antibodies. Specifically, the method comprises testing a sample from a human, having undergone at least one course of immunotherapy, to measure the level of therapeutic anti-PD-1 and/or antj-PD-L1 antibodies, wherein depending on the level of the antibodies, further treatment with the antibodies can be adjusted. In a further embodiment, the disclosure herein relates to a method for detecting the responsiveness of a patient to a therapeutic antibody treatment, wherein the therapeutic antibody is directed against PD-1 and/or PD-L1 , the method comprising determining, in a sample from the patient, the presence or amount of inactive TNFalpha, said presence or amount being indicative of low or decreasing responsiveness of the patient to said treatment In a preferred embodiment, the method also comprises a determination of the presence or amount of the therapeutic antibody and Anti-Drug Antibodies directed against said therapeutic antibody to obtain a patient profile, wherein the patient profile indicates the responsiveness to said treatment.

PP-1 and PIH1

The present invention relates to fusion proteins comprising an extracellular domain of PD-1 (programmed cell death- 1 protein) and an oligomerizatbn domain, and/or to fusion proteins comprising an extracellular domain of PD-L1 (programmed cell death-ligand 1 protein) and an oligomerization domain. The PD-1 and PD-L1 sequences may be variants of the native human sequences. As used herein, the term "variant" refers to an entity that shows significant structural identity with a reference entity but differs structurally from the reference entity in the presence or level of one or more chemical moieties as compared with the reference entity. In many embodiments, a variant also differs functionally from its reference entity. In general, whether a particular entity is properly considered to be a "variant" of a reference entity is based on its degree of structural identity with the reference entity. As will be appreciated by those skilled in the art, any biological or chemical reference entity has certain characteristic structural elements. A variant, by definition, is a distinct chemical entity that shares one or more such characteristic structural elements. A polypeptide may have a characteristic sequence element comprised of a plurality of amino acids having designated positions relative to one another in linear or three- dimensional space and/or contributing to a particular biological function. For example, a variant polypeptide may differ from a reference polypeptide as a result of one or more differences in amino acid sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, etc) covalently attached to the polypeptide backbone. In some embodiments, a variant polypeptide shows an overall sequence identity with a reference polypeptide that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. Alternatively or additionally, in some embodiments, a variant polypeptide does not share at least one characteristic sequence element with a reference polypeptide. In some embodiments, the reference polypeptide has one or more biological activities. In some embodiments, a variant polypeptide shares one or more of the biological activities of the reference polypeptide. In some embodiments, a variant polypeptide lacks one or more of the biological activities of the reference polypeptide. In some embodiments, a variant polypeptide shows a reduced level of one or more biological activities as compared with the reference polypeptide. In many embodiments, a polypeptide of interest is considered to be a "variant" of a parent or reference polypeptide if the polypeptide of interest has an amino acid sequence that is identical to that of the parent but for a small number of sequence alterations at particular positions. Typically, fewer than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% of the residues in the variant are substituted as compared with the parent In some embodiments, a variant has 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substituted residue as compared with a parent. Often, a variant has a very small number (e.g., fewer than 5, 4, 3, 2, or 1 ) number of substituted functional residues (i.e., residues that participate in a particular biological activity). Furthermore, a variant typically has not more than 5, 4, 3, 2, or 1 additions or deletions, and often has no additions or deletions, as compared with the parent Moreover, any additions or deletions are typically fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and commonly are fewer than about 5, about 4, about 3, or about 2 residues. In some embodiments, the parent or reference polypeptide is one found in nature. As will be understood by those of ordinary skill in the ait, a plurality of variants of a particular polypeptide of interest may commonly be found in nature, particularly when the polypeptide of interest is an infectious agent polypeptide. In one embodiment described herein, the fusion protein comprises the predicted extracellular domain of PD-1 protein (SEQ ID NO: 1) or a fragment thereof. In one aspect, the extracellular domain of PD-1 protein comprises SEQ ID NO: 12 or a fragment thereof. In another aspect, the extracellular domain of PD-1 may have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the polypeptide sequence consisting of the amino acids at positions 21 to 170 of SEQ ID NO: 1.

The extracellular domain of PD-1 comprises a polypeptide comprising residues 1 to 171 of SEQ ID NO: 1 , a polypeptide comprising residues 1 to 170 of SEQ ID NO: 1 , a polypeptide comprising residues 1 to 145 of SEQ ID NO: 1, a polypeptide comprising residues 21 to 171 of SEQ ID NO: 1 , a polypeptide comprising residues 21 to 170 of SEQ ID NO: 1, a polypeptide comprising residues 21 to 145 of SEQ NO: 1 , a polypeptide comprising residues 33 to 171 of SEQ ID NO: 1, a polypeptide comprising residues 33 to 170 of SEQ ID NO: 1, a polypeptide comprising residues 33 to 145 of SEQ ID NO: 1, a polypeptide comprising residues 35 to 171 of SEQ ID NO: 1 , a polypeptide comprising residues 35 to 170 of SEQ ID NO: 1, and a polypeptide comprising residues 35 to 145 of SEQ ID NO: 1. In one embodiment the extracellular domain of PD-1 may have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to a polypeptide comprising residues 1 to 171 of SEQ ID NO: 1, a polypeptide comprising residues 1 to 170 of SEQ ID NO: 1, a polypeptide comprising residues 1 to 145 of SEQ ID NO: 1 , a polypeptide comprising residues 21 to 171 of SEQ ID NO: 1 , a polypeptide comprising residues 21 to 170 of SEQ ID NO: 1, a polypeptide comprising residues 21 to 145 of SEQ NO: 1 , a polypeptide comprising residues 33 to 171 of SEQ ID NO: 1 , a polypeptide comprising residues 33 to 170 of SEQ ID NO: 1, a polypeptide comprising residues 33 to 145 of SEQ ID NO: 1, a polypeptide comprising residues 35 to 171 of SEQ ID NO: 1 , a polypeptide comprising residues 35 to 170 of SEQ ID NO: 1 , and a polypeptide comprising residues 35 to 145 of SEQ ID NO: 1. In yet another embodiment the extracellular domain of PD-1 comprises the amino acid sequence set forth in SEEQ ID NO: 12. In another embodiment, the extracellular domain of PD-1 may have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence set forth in SEQ ID NO: 12.

In another embodiment described herein, the fusion protein comprises the extracellular domain of PD-L1 protein (SEEQ ID NO: 2) or a fragment thereof. In another embodiment, the extracellular domain of PD-L1 protein comprises SEEQ ID NO: 13 or a fragment thereof. In addition, the extracellular domain of PD-L1 may have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the polypeptide sequence consisting of the amino acids at positions 19 to 238 of SEEQ ID NO: 2.

The extracellular domain of PD-L1 may be a polypeptide comprising residues 1 to 240 of SEQ ID NO: 2, a polypeptide comprising residues 1 to 239 of SEEQ ID NO: 2, a polypeptide comprising residues 1 to 238 of SEEQ ID NO: 2, a polypeptide comprising residues 19 to 240 of SEEQ ID NO: 2, a polypeptide comprising residues 19 to 239 of SEEQ ID NO: 2, a polypeptide comprising residues 19 to 238 of SEEQ NO: 2, a polypeptide comprising residues 18 to 239 of SEEQ ID NO: 2, a polypeptide comprising residues

18 to 238 of SEEQ ID NO: 2, a polypeptide comprising residues 18 to 225 of SEQ ID NO: 2, a polypeptide comprising residues 18 to 134 of SEEQ ID NO: 2, a polypeptide comprising residues 18 to 127 of SEQ ID NO: 2, a polypeptide comprising residues 19 to 239 of SEQ ID NO: 2, a polypeptide comprising residues

19 to 238 of SEQ ID NO: 2, a polypeptide comprising residues 19 to 225 of SEQ ID NO: 2, a polypeptide comprising residues 19 to 134 of SEQ ID NO: 2, a polypeptide comprising residues 19 to 127 of SEQ ID NO: 2, a polypeptide comprising residues 133 to 225 of SEQ ID NO: 2, a polypeptide comprising residues 133 to 238 of SEQ ID NO: 2, and a polypeptide comprising residues 133 to 239 of SEQ ID NO: 2. In one embodiment, the extracellular domain of PD-L1 may have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to a polypeptide comprising residues 1 to 240 of SEQ ID NO: 2, a polypeptide comprising residues 1 to 239 of SEQ ID NO: 2, a polypeptide comprising residues 1 to 238 of SEQ ID NO: 2, a polypeptide comprising residues 19 to 240 of SEQ ID NO: 2, a polypeptide comprising residues 19 to 239 of SEQ ID NO: 2, a polypeptide comprising residues 19 to 238 of SEQ NO: 2, a polypeptide comprising residues 18 to 239 of SEQ ID NO: 2, a polypeptide comprising residues 18 to 238 of SEQ ID NO: 2, a polypeptide comprising residues 18 to 225 of SEQ ID NO: 2, a polypeptide comprising residues 18 to 134 of SEQ ID NO: 2, a polypeptide comprising residues 18 to 127 of SEQ ID NO: 2, a polypeptide comprising residues 19 to 239 of SEQ ID NO: 2, a polypeptide comprising residues 19 to 238 of SEQ ID NO: 2, a polypeptide comprising residues 19 to 225 of SEQ ID NO: 2, a polypeptide comprising residues 19 to 134 of SEQ ID NO: 2, a polypeptide comprising residues 19 to 127 of SEQ ID NO: 2, a polypeptide comprising residues 133 to 225 of SEQ ID NO: 2, a polypeptide comprising residues 133 to 238 of SEQ ID NO: 2, and a polypeptide comprising residues 133 to 239 of SEQ ID NO: 2. In yet another embodiment, the extracellular domain of PD-L1 comprises the amino acid sequence set forth in SEQ ID NO: 13. In another embodiment, the extracellular domain of PD-L1 may have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence set forth in SEQ ID NO: 13. In addition, certain residues may be modified, by site-directed-mutagenesis, for improved higher affinity interaction between known mAbs and their target In some embodiments, the modification constitutes substitution of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residue as compared with a parent Often, modification utilizing site-directed-mutagenesis has a very small number (e.g., fewer than 5, 4, 3, 2, or 1) of substituted functional residues (i.e., residues that participate in a binding affinity). Furthermore, modification utilizing site-directed-mutagenesis typically has not more than 5, 4, 3, 2, or 1 additions or deletions, and often has no additions or deletions, as compared with the parent. Moreover, any additions, deletions or substitutions are typically fewer than about 50 residues, about 45 residues, about 40 residues, about 35 residues, about 30 residues, about 25 residues, about 20 residues, about 19 residues, about 18 residues, about 17 residues, about 16 residues, about 15 residues, about 14 residues, about 13 residues, about 12 residues, about 11 residues, about 10 residues, about 9 residues, about 8 residues, about 7 residues, about 6 residues, and commonly are fewer than about 5 residues, about 4 residues, about 3 residues, or about 2 residues. In some embodiments, the parent or reference polypeptide is one found in nature.

In one embodiment, one or more amino acid residues) within PD-1 critical for interaction with an anti-PD- 1 antibody is/are modified for improved higher affinity interaction between antibodies and their target. In some embodiment, the one or more amino acid residues) modified within PD-1 are involved in hydrogen bonding. In another embodiment the one or more amino acid residue(s) modified within PD-1 are involved in forming salt bridges. In yet another embodiment, the one or more amino acid residues) modified within PD-1 are involved in water mediated interactions. In yet another embodiment, the one or more amino acid residues) modified within PD-1 are involved in van der Waals interactions.

In one embodiment one or more amino acid residue(s) within PD-1 critical for interaction with the Pembrolizumab mAb is/are modified. In a related embodiment, the one or more amino acid residue(s) modified within PD-1 can be selected from the group consisting of PD-IN66, PD-IT76, PD-IK78, PD-IS87, _pd . IK131 PD-IF63, PD-IE84, PD-IS87, _PD -IG90 and _PD -iA132. In another related embodiment, the amino acid residue modified within PD-1 is PD-ID85. In yet another embodiment, related embodiment, the one or more amino acid residues) modified within PD-1 can be selected from the group consisting of PD-IK78, PD-IS87 and _PD -iD85. In yet another embodiment related embodiment, the one or more amino acid residues) modified within PD-1 can be selected from the group consisting of PD-IV64, PD-IY68, PD-IQ75, PD-ID77, PD-1A8I, P&.1F82, PD-iP83, P&.1R86, PD-1Q88, PD-iP89, PD-IG90, PD-IM26, PD-IL128, PD-IA129, and PD-11134. In yet another embodiment, related embodiment, the one or more amino acid residue(s) modified within PD-1 can be selected from the group consisting of PD-IN66, PD-IT76, PD-IK78, PD-IS87, _PD - |K131 PD-IF63, PD-IE84, PD-IS87, PD-IG90, PD-IA132, PD-ID85, PD-IK78, PD-IS87 and PD-ID85, PD-IV64, _PD - 1Y68, PD-iQ.75, PD-1D77, PD-1A8I, PD-iF82, PD-iP83, PD.1R86, PD-1Q88, PD-iP89, PD-iG90, pen 1126, PD-IL128, PD- ₁ A129, and _PD -il134.

In one embodiment one or more amino acid residues) within PD-1 critical for interaction with the Nivolumab mAb is/are modified. In a related embodiment, the one or more amino acid residues) modified within PD-1 can be selected from the group consisting of PD-IP28, PD-IL128, _P D-IA129, _PD -IP130, PD-1 A132, PD-iD29, PD-iR30, PD-1S6O and _PD -IK131. In another related embodiment the one or more amino acid residue(s) modified within PD-1 can be selected from the group consisting of PD-IS27, PD-IP28, PD- IP31 , PD-1E6I , PD-iA129, PD-iP130, PD-iK131, PD-iA132 and PD-IQ133.

In one embodiment, the one or more amino acid residues) modified within PD-1 are involved in hydrogen bonding or van der waals interactions between either Nivolumab or Pembrolizumab and PD-1. In a specific embodiment, the one or more amino acid residues) modified within PD-1 can be selected from the group consisting of PD-iL.128, pD-iA129, pD-iA130, pD-iK131 , and P_MA132.

In one embodiment, one or more amino acid residues) within the Pembrolizumab mAb critical for interaction with PD-1 is/are modified. In some embodiments, the one or more amino acid residues) within the Pembrolizumab mAb that are modified include residues within the heavy chain CDR1 (HCDR1 ). In some embodiments, the one or more amino acid residue(s) modified within the HCDR1 Pembrolizumab mAb can be selected from the group consisting of heavyT30, heavy Y33, and heavy Y35. In some embodiments, the one or more amino acid residues) within the Pembrolizumab mAb that are modified include residues within the heavy chain CDR2 (HCDR2). In some embodiments, the one or more amino acid residue(s) modified within the HCDR2 Pembrolizumab mAb can be selected from the group consisting of heavyN52, hoavyS54, heavyN55, heavyG57, hoavyT58, and hoavyN59. In some embodiments, the one or more amino acid residues) within the Pembrolizumab mAb that are modified include residues within the heavy chain CDR3 (HCDR3). In some embodiments, the one or more amino acid residues) modified within the HCDR3 Pembrolizumab mAb can be selected from the group consisting of h∞vyR99, heavy Y101 , heavyR102, heavyF103, heavyD104, heavyM105, and heavyD108. In some embodiments, the one or more amino acid residues) within the Pembrolizumab mAb that are modified include residues within the light chain CDR1 (LCDR1 ). In some embodiments, the one or more amino acid residues) modified within the LCDR1 Pembrolizumab mAb can be selected from the group consisting of ightT31 , iightS32, » _8Μ Υ34, and iightY36. In some embodiments, the one or more amino acid residues) within the Pembrolizumab mAb that are modified include residues within the light chain CDR2 (LCDR2). In some embodiments, the one or more amino acid residues) modified within the LCDR2 Pembrolizumab mAb can be selected from the group consisting of ightY53, iightL54, ι _¾Μ Υ57, and iightE5S. In some embodiments, the one or more amino acid residues) within the Pembrolizumab mAb that are modified include residues within the light chain CDR3 (LCDR3). In some embodiments, the one or more amino acid residue(s) modified within the LCDR3 Pembrolizumab mAb can be selected from the group consisting of iightS95, BghtR96, iightD97, iightl-98 and BghtL.100.

In one embodiment, one or more amino acid residues) within the Nivolumab mAb that critical for interaction with PD-1 is/are modified. In some embodiments, the one or more amino acid residues) within the Nivolumab mAb that are modified include residues within the heavy chain CDR1 (HCDR1). In some embodiments, the one or more amino acid residues) modified within the HCDR1 Pembrolizumab mAb can be selected from the group consisting of heavyG26, h«vyl27, heavy N31 and h∞vyG33. In some embodiments, the one or more amino acid residue(s) within the Nivolumab mAb that are modified include residues within the heavy chain CDR2 (HCDR2). In some embodiments, the one or more amino acid residue(s) modified within the HCDR2 Pembrolizumab mAb can be selected from the group consisting of heavyV50, hoavyW52 and heavy Y53. In some embodiments, the one or more amino acid residues) within the Nivolumab mAb that are modified include residues within the heavy chain CDR3 (HCDR3). In some embodiments, the one or more amino acid residue(s) modified within the HCDR3 Pembrolizumab mAb can be selected from the group consisting of h∞vyN99, ho_vyD100, heavyD101 and h∞vyY102. In some embodiments, the one or more amino acid residues) within the Nivolumab mAb that are modified include residues within the light chain CDR1 (LCDR1 ). In some embodiments, the one or more amino acid residue(s) within the Nivolumab mAb that are modified include residues within the light chain CDR2 (LCDR2). In some embodiments, the one or more amino acid residues) modified within the LCDR2 Pembrolizumab mAb can be selected from the group consisting of iightL46, light A55 and iightT56. In some embodiments, the one or more amino acid residue(s) within the Nivolumab mAb that are modified include residues within the light chain CDR3 (LCDR3). In some embodiments, the amino acid residue modified within the LCDR3 Pembrolizumab mAb is »ghtS91.

In another embodiment, one or more amino acid residues) within PD-L1 critical for interaction with an anti-PD-L1 antibody is/are modified for improved higher affinity interaction between antibodies and their target In some embodiment, the one or more amino acid residue(s) modified within PD-L1 are involved in hydrogen bonding. In another embodiment, the one or more amino acid residues) modified within PD- L1 are involved in forming salt bridges. In yet another embodiment, the one or more amino acid residue(s) modified within PD-L1 are involved in water mediated interactions. In yet another embodiment, the one or more amino acid residues) modified within PD-L1 are involved in van der Waals interactions. Ollgomerization domains

The present invention discloses fusion proteins comprising an extracellular domain of PD-1 (programmed cell death- 1 protein) or an extracellular domain of PD-L1 (programmed cell death-ligand 1 protein) and an oligomerization domain. The oligomerization domain described herein may comprise a domain selected from the group consisting of a Fc domain, a Clathrin trimerization domain, a GCN4 trimerization domain, a p53 tetramerization domain, and pentameric domains or even a hingeless monomelic Fc domain creating novel proteins. In some embodiments, the oligomerization domain lacks the oligomerization region and the fusion protein is a monomer. In some embodiments, the oligomerization domain is a hingeless monomeric Fc domain and the fusion protein is a monomer. The oligomerization domain may comprise virus like particles as a structure for exposure of the antigen or antibody variable domains. While these oligomerization domains are known by those expert in the art, they have not been used to create novel proteins for diagnostics.

In some aspects, the oligomerization domain may comprise an amino acid sequence selected from the group consisting of an Fc domain, a fragment of an Fc domain, and a variant of an Fc domain. In one embodiment, the fusion protein described herein can be part of a higher order structure, such as a protein or multimeric complex. In some embodiments, the PD-1 and/or PD-L1 protein is fused to the Fc domain, or fragment thereof, via one or more peptide linkers. In one embodiment, the PD-1 and/or PD-L1 protein is fused to the C-terminus of said Fc domain, or fragment thereof. In another embodiment, the PD-1 and/or PD-L1 protein is fused to the N-terminus of said Fc domain, or fragment thereof. In some embodiments, the oligomerization domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 28. In one embodiment, the oligomerization domain is selected from the group consisting of the amino acid sequence set forth in SEQ ID NO: 9 (GCN4 trimer domain), the amino acid sequence set forth in SEQ ID NO: 10 (clathrin trimer domain), and the amino acid sequence set forth in SEQ ID NO: 11 (p53 tetramer domain). In some embodiments, the Fc domain may have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence set forth in SEQ ID NO: 9 (GCN4 trimer domain). In some embodiments, the Fc domain may have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence set forth in SEQ ID NO: 10(clathrin trimer domain). In some embodiments, the Fc domain may have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence set forth in SEQ ID NO: 11 (p53 tetramer domain).

In one embodiment, the Fc domain comprises an immunoglobulin CH2 domain or an immunoglobulin CH3 domain. In another embodiment, the Fc domain comprises an immunoglobulin CH2 and CH3 domain. In some embodiments, the Fc domain is selected from the group consisting of lgG1 CH2 and CH3 domain, lgG4 CH2 and CH3 domain, lgG1 CH2 and an lgG4 CH3 domain, and lgG4 CH2 and an lgG1 CH3 domain. In some embodiments, the Fc domain may be of mouse or human. In some embodiments, the Fc domain may be of mouse lgG1 or lgG2A. In other embodiments, the Fc domain comprises an IgG hinge domain.

In one embodiment, the Fc domain is selected from the group consisting of the amino acid sequence set forth in SEQ ID NO: 6 (murine lgG1 , CH2 and CH3), the amino acid sequence set forth in SEQ ID NO: 7 (murine lgG1 Fc), the amino acid sequence set forth in SEQ ID NO: 8 (murine lgG2A Fc), and the amino acid sequence set forth in SEQ ID NO: 28 (murine lgG2A, CH2 and CH3). In some embodiments, the Fc domain may have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the Fc domain may have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the Fc domain may have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the Fc domain may have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence set forth in SEQ ID NO: 28.

As used herein, the term Ig "hinge" region refers to a polypeptide comprising an amino acid sequence that shares sequence identity, or similarity, with a portion of a naturally-occurring Ig hinge region sequence, which includes the cysteine residues at which the disulfide bonds link the two heavy chains of the immunoglobulin. Sequence similarity of the hinge region linkers described herein with naturally- occurring immunoglobulin hinge region amino acid sequences can range from at least 50% to about 75- 80%, and typically greater than about 90%. In some embodiments, the murine lgG1 Fc domain of the fusion protein of the present application comprises a hinge. In some embodiments, the murine lgG1 Fc domain of the fusion protein of the present application lacks a hinge. Exemplary amino acid sequence of murine lgG1 Fc domains are SEQ ID NO:6 (without hinge) and SEQ ID NO:7 (with hinge).

In some embodiments, the murine lgG2A Fc domain of the fusion protein of the present application comprises a hinge. In some embodiments, the murine lgG2A Fc domain of the fusion protein of the present application lacks a hinge. Exemplary amino acid sequence of murine lgG2A Fc domains are SEQ ID NO:28 (without hinge) and SEQ ID NO:8 (with hinge).

Llnkfifs

In some embodiments, the PD-1 and/or PD-L1 protein is fused to the oligomerization domain, or fragment thereof, via one or more peptide linkers. In one embodiment, the PD-1 and/or PD-L1 protein is fused to the C-terminus of said oligomerization domain, or fragment thereof, via one or more peptide linkers. In another embodiment, the PD-1 and/or PD-L1 protein is fused to the N-terminus of said oligomerization domain, or fragment thereof, via one or more peptide linkers.

In some embodiments, a peptide linker may consist of a sequence of consecutive amino acids that typically include at least one glycine, and at least one serine. A peptide linker may also consist of a sequence of consecutive amino acids that typically include at least one glycine, at least one proline and at least one serine. Exemplary flexible linkers and flexible pro linkers include the amino acid sequences set forth in SEQ ID NO:4 (GGGS) or SEQ ID NO:5 (GGGPS), although the precise amino acid sequence of an peptide linker is not particularly limiting. In some embodiments, the PD-1 and/or PD-L1 protein is fused to the oligomerization domain, or fragment thereof, via two or more different peptide linkers. In some embodiments the PD-1 and/or PD-L1 protein is fused to the oligomerization domain, or fragment thereof, via two or more identical peptide linkers

A peptide linker, as described herein, is typically not found in nature, although some naturally-occurring amino acid sequences may serve as suitable linkers or may be used to design suitable linkers. A linker may include about 1 to 30 amino acids, such as about 2 to 25, about 3 to 20, about 4 to 16, or about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

Signal Peptide Sequences

Fusion proteins disclosed herein typically comprise a signal peptide sequence (or "signal sequence" to favor translocation of the protein across the cell membrane of an expression vector, such as a mammalian cell, especially a human cell. A polypeptide may nevertheless lack a signal peptide sequence, for example, if the fusion protein disclosed herein typically comprise a signal peptide sequence to favor translocation of the polypeptide across the cell membrane of an expression vector, such as a mammalian cell, especially a human cell. A fusion protein may nevertheless lack a signal peptide sequence. Synthetically-produced polypeptides may similarly lack a signal peptide sequence. A signal peptide sequence is typically included at the N-terminus of a fusion protein. A signal peptide sequence is preferably sufficient to translocate the protein outside of the cell surface membrane of a eukaryotic cell (e.g., a mammalian cell, such as a human cell) following the translation of the protein in the eukaryotic cell, although other sequence motifs of a fusion protein may also aid translocation.

An exemplary signal peptide sequence has the amino acid sequence set forth in SEQ ID NO:3 (MYRMQLLSCIALSLALVTNS), which is the human interleukin-2 signal peptide sequence. This well- characterized sequence is capable of translocating protein out of both human cells and other mammalian cells.

Affinity Taos

A fusion protein may optionally include an affinity tag. Affinity tags are useful for purification, and they may also be useful in assays that utilize a fusion protein. Exemplary affinity tags include polyhistidine, chitin binding protein, maltose binding protein, Strep-tag, glutathione-S-transferase, FLAG-tag, V5-tag, Myc-tag, HA-tag, NE-tag, AviTag, Calmodulin-tag, polyglutamate, S-tag, SBP-tag, Softag 1, Softag 3, TC tag, VSV-tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, biotjn carboxyl carrier protein, green fluorescent protein-tag, HaloTag, Nus-tag, and thioredoxin-tag, although the choice of affinity tag is not particularly limiting. A fusion protein may nevertheless lack an affinity tag, for example, if the affinity tag is removed after use or if the fusion protein is purified using a strategy that does not require an affinity tag. An exemplary affinity tag is polyhistidine, which typically includes an amino acid sequence comprising seven consecutive histidines (SEQ ID NO:31). Another exemplary affinity tag is a polyhistidine tag comprising between 4 and 10 consecutive histidines. Fusion proteins

The fusion proteins featured in the embodiments described herein may comprise an extracellular domain of PD-1 (programmed cell death- 1 protein) or an extracellular domain of PD-L1 (programmed cell death- ligand 1 protein) and an oligomerization domain, wherein the oligomerization domain is selected from the group consisting of a murine lgG1 Fc domain, a murine lgG2A Fc domain, a GCN4 trimer domain, a clathrin trimer domain, and a p53 tetramer domain, or a fragment thereof.

A fusion protein of the sort disclosed herein may have the amino acid sequence set forth in SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:30. A fusion protein may have an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17. SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21 , SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:30. ΝμςΙθΙς Aeld¾ Cloning 0?»!?, ?nd Expr^lpn Cell?

Embodiments described herein also include a nucleic acid comprising a nucleotide sequence encoding a fusion protein described herein. The nucleic acid may be DNA or RNA. DNA comprising a nucleotide sequence encoding a fusion protein described herein typically comprises a promoter that is operably- linked to the nucleotide sequence. The promoter is preferably capable of driving constitutive or inducible expression of the nucleotide sequence in an expression cell of interest. The precise nucleotide sequence of the nucleic acid is not particularly limiting so long as the nucleotide sequence encodes a fusion protein described herein. Codons may be selected, for example, to match the codon bias of an expression cell of interest (e.g., a mammalian cell such as a human cell) and/or for convenience during cloning. DNA may be a plasmid, for example, which may comprise an origin of replication (e.g., for replication of the plasmid in a prokaryotic cell).

Various aspects of the present invention also relate to a cell comprising a nucleic acid comprising a nucleotide sequence that encodes a fusion protein as described herein. The cell may be an expression cell or a cloning cell. Nucleic acids are typically cloned in E∞li, although other cloning cells may be used. If the cell is an expression cell, the nucleic acid is optionally a nucleic acid of a chromosome, i.e., wherein the nucleotide sequence is integrated into the chromosome, although then nucleic acid may be present in an expression cell, for example, as extrachromosomal DNA.

Various aspects of the present invention include a cell comprising a nucleic acid comprising a sequence that encodes a fusion protein as described herein. The cell is typically an expression cell. The nature of the expression cell is not particularly limiting. Mammalian expression cells may allow for favorable folding, post-translational modifications, and/or secretion of a fusion protein or oligomeric fusion protein, although other eukaryotic cells or prokaryotic cells may be used as expression cells. Exemplary expression cells include CHO, BHK, NS0, Sp2/0, COS, C127, HEK, HT-1080, PER.C6, HeLa, and Jurkat cells.

Another aspect of the present invention herein provides a method of making a fusion protein comprising an extracellular domain of PD-1 (programmed cell death- 1 protein) and/or an extracellular domain of PD- L1 (programmed cell death-ligand 1 protein), and an oligomerization domain comprising the steps of: a) transfecting a host cell with a nucleic acid molecule encoding the fusion protein according to the present invention, b) making the fusion protein by expressing the nucleic acid molecule of () in the host cell.

Immunoassays and reagents Immunoassays are usually used to measure cell surface antigens or to measure antibodies in a sample. Typically, immunofluorescence using flow cytometry is the immunoassay of choice. However, other immunoassays may be used, for example enzyme linked immunosorbent assays (ELISA). This technique is based upon the special properties of antigen-antibody interactions with simple phase separations to produce powerful assays for detecting biological molecules.

The methodology and instrumentation for the ELISA is simpler than that for immunofluorescence. Yet further, the ELISA and immunofluorescence assays are completely different assays. ELISA assays measure the protein (antigen) or antibody in the plasma/serum, which reflects the entire body. On the contrary, surface immunofluorescent assays specifically measure an antigen on the surface of individual cells and do not provide information on the amount of cells in the body. Thus, there are advantages in developing an ELISA assay to provide a measurement of the entire body.

One well-known and highly specific ELISA is a sandwich ELISA. In this assay, the antibody is bound to the solid phase or support, which is then contacted with the sample being tested to extract the antigen from the sample by formation of a binary solid phase antibody:antigen complex. After a suitable incubation period, the solid support is washed to remove the residue of the fluid sample and then contacted with a solution containing a known quantity of labeled antibody.

Some embodiments of the present invention are related to immunoassays for assaying a composition containing anti-PD-1 and/or antj-PD-L1 antibodies. Other embodiments relate to immunoassays for assaying a composition containing PD-1 and/or PD-L1 antigens.

Exemplary immunoassays according to the present invention are, for example, ELISA assays (Enzyme- Linked Immunosorbent Assay), which uses either colorimetric, fluorescent or chemiluminescent detection. Other exemplary immunoassays according to the present invention are well known by the skilled person, e.g. ELISA Using Slope Correction, RIA (radioimmunoassay), competitive EIA (competitive enzyme immunoassay), DAS-ELISA (double antibody sandwich-ELISA), bridging-ELISA, techniques based on the use of protein or antibody mbroarrays (including liquid bead-based arrays like those based on Luminex technology), technologies based on discrete microparticles, assays based on the precipitation of colloidal gold or other visible microparticles or fluorescent particles like lateral or vertical flow tests based on immunochromatography typically used for rapid testing and Point of Care applications using either visual (by eye) interpretation or a reader like a smartphone or a portable reader or a benchtop reader, affinity chromatography techniques, Homogeneous Mobility Shift Assays (HMSA), ligand binding assays, lectin binding assays, biosensors, etc.

It is known that the circulating anti-PD-1 antibodies have a similar half-life to other lgG4 antibodies (Farolfi et al 2016). Also, the PD-1 antibodies coat circulating T cells and effectively remove themselves from circulation (are no longer freely circulating in plasma). This causes a relatively rapid decline in circulating anti-PD-1 (nivolumab) levels in humans. The relationship between circulating levels, T-cell resident levels and outcome are not clear and thus an assay which can shed light on this is valuable. However, it is known that anti-PD-1 (Nivolumab) remains resident on circulating T cells for an extended period with an occupancy level of 60-80% depending upon the initial dose at 0.3 to 10 mg/kg (Brahmer et al 2010). In this function anti-PD-1 act as an antagonist of PD-1 preventing ligand binding to PD-L1 on tumors and the subsequent shut down of the immune response. It will be important to correlate dosing, T cell occupancy and measurable circulating anti-PD-1 or antj PD-L1 with outcomes. It is, therefore, an objective of the present disclosure to provide methods for detecting and/or monitoring anti-PD-1 and/or anti-PD-L1 antibodies.

Specifically, anti-PD-1 and/or anti-PD-L1 therapeutic antibodies, antibody:antigen complexes and/or antibodies that are directed at PD-1 and/or PD-L1 may be detected or monitored by using a modified sandwich ELISA technique or other immunoassay technique known to those familiar in the art.

In a particular embodiment, the concentration of the anti-PD-1 or anti-PD-L1 biological drugs is measured by ELISA, similarly that it is shown in the examples of the present invention.

The fusion proteins described herein may also be used to prevent or compete for binding of antibody to the same antigens on the surface of T cells or upon tumor cells.

In one aspect, anti-PD-1 and/or anti-PD-L1 therapeutic antibodies are measured in a patient that has undergone at least one dose of immunotherapy with a therapeutic antibody. The therapeutic antibody may include, but is not limited to anti-PD-1 or anti-PD-L1 therapeutic antibodies. The antibody:antigen complex is measured by ELISA techniques or other immunoassays and provides a determination of the circulating level of the therapeutic antibody. The interpretation of this result and the changes in treatment are then determined by an oncologist familiar with the clinical signs and symptoms to determine efficacy of the antibody immunotherapy. In some patients antibody treatments are cleared faster than in others, in particular if patients have different allotypes (constant region polymorphisms).

One aspect of the present invention includes a method of providing an immunotherapy to a patient comprising administering to the patient a therapeutic antibody and detecting the presence of anti-PD-1 and/or anti-PD-L1 therapeutic antibodies using the fusion proteins described herein. It is envisioned that these methods can be used to monitor the efficacy of antibody-based therapy. In one embodiment, a physician may wish to increase dose if efficacy is slow to appear and is not removing symptoms. These tools we describe herein will allow sensitive detection of the expected change in circulating levels of therapeutic antibody.

In further embodiments, the present invention provides methods for detecting and/or monitoring diseases mediated by PD-1 and/or PD-L1 by measuring anti-PD-1 and/or antj-PD-L1 therapeutic antibodies. Said methods for detecting and/or monitoring diseases mediated by PD-1 and/or PD-L1 comprises contacting a sample from a patient with a fusion protein described herein. In further embodiments, the methods for detecting and/or monitoring diseases mediated by PD-1 and/or PD-L1 is conducted on a patient that has undergone a course of immunotherapy with a therapeutic antibody.

Some aspects of the present invention relate to a method of determining the amount of circulating levels of a biotherapeutic antibody selected from the group consisting of nivolumab, pembrolizumab and other anti-PD-1 therapies. In some embodiments, the method of determining the amount of circulating levels of a biotherapeutic antibody comprise the steps of (i) obtaining a sample from a patient undergoing the antibody treatment, and (ii) contacting the sample with the fusion protein according to the present invention. Other aspects of the present invention relate to a method of determining the amount of (free) circulating levels of a biotherapeutic antibody selected from the group consisting of Atezolizumab, Avelumab, and other anti-PD-L1 therapies, comprising (i) obtaining a sample from a patient undergoing the antibody treatment, and (ii) contacting the sample with the fusion protein according to the present invention.

Some embodiments are related to a composition comprising a fusion protein comprising PD-1 and/or PD- L1 , or a fragment thereof, and an oligomerization domain. Further provided herein is a method of making a composition comprising a fusion protein comprising PD-1 and/or PD-L1 or a fragment thereof and an oligomerization domain as described herein. A kit comprising the fusion protein or the composition is also provided.

Some embodiments include an immunoassay reagent comprising a fusion protein as described herein for assaying a composition containing anti-PD-1 and/or anti-PD-L1 antibodies. The immunoassay reagent may be bound to a solid support. A solid support may be, for example, a bead, membrane, microtiter plate, polypeptide chip, or the solid-phase of a chromatography column.

Various embodiments also include a device for assaying a composition containing anti-PD-1 and/or anti- PD-L1 antibodies. The device may be a device for determining whether a sample contains anti-PD-1 and/or anti-PD-L1 antibodies. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used and will be apparent to those of skill in the art. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.

Throughout this specification and claims, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of this invention. All literature and patent citations mentioned herein are expressly incorporated by reference.

EXAMPLES

Example 1 : Expression and purification of checkpoint proteins Genes encoding the various fusion protein of human PD-1 and PD-L1 were synthetically produced, codon optimized for mammalian expression, and cloned into either pTT5 vector or other high expressing mammalian vector. Low endotoxin scale-up preparations of plasmid DNA were performed to prepare for transient mammalian expression. Cultures of Human embryonic kidney (HEX) 293 derivative cells were transiently transfected with scaled- up expression plasmids and grown, utilizing standard transection protocol. Cell pellets were harvested by centrifugation and protein expression in the cell culture supernatant (CCS) was verified by western blot and SDS-PAGE 4-5 days post-transfection.

Fusion proteins comprising lgG1 or lgG2a were purified by Protein A affinity chromatography and formulated via dialysis, aliquoted, and stored at -80°C. Proteins containing the p53 multimerization domains had a C-terminal histidine tag and were purified by immobilized metal affinity chromatography, buffer exchanged, aliquoted, and stored at -80°C. Protein expression and confirmation of tetramerization was assessed by SDS-PAGE, and CE-SDS (PD-1-p53-His).

Figures 1 and 2 show 4-20% tris-glycine TGX™ SDS-PAGE gels run under reducing and non-reducing conditions, respectively. The gels were stained with the InstantBlue™ protein stain. The lanes of each gel are numbered 1 to 10 from left to right. The first lane contains a molecular weight marker, lanes 2-5 contain fusion proteins corresponding to SEQ ID NO: 14 to 17 and lanes 6 to 8 contain fusion proteins corresponding to SEQ ID NO: 21 to 23, which were cloned and then expressed in eukaryotic cells. Each fusion protein was capable of expression at a detectable level. Example 2: Specificity of PD-1 and PD-L1 recombinant proteins

2a. Specific reactivity against anti-PD-1 and anti-PD-L1 antibodies

Several recombinant PD-1 and PD-L1 proteins were tested for their specific reactivity against anti-PD-1 and anti-PD-L1 antibodies from commercial sources. Recombinant versions of PD-L1 and PD-1 proteins were tested separately with directly labeled fluorescent antibodies using ELISA assays.

ELISA plates were coated with PD-1 or PD-L1 fusion proteins and detected with APC conjugated anti- PD-1 or anti-PDL-1 antibodies using a fluorescent reader. The different antigens and different versions of the same antigen were tested at equimolar concentrations, 50nM to compensate for the different MW of the proteins. FIG. 3A shows specific reactivity of PD-1-p53 (PD1-(GGGPS)2-p53-HisTag, SEQ ID N029) against anti-PD-1 antibody in a serial dilution series (dilutions from 0.5ug/ml to 0.04u.g/ml). FIG. 3B shows specific reactivity of PD-L1-p53 proteins (PD-L1-(GGGPS)2-p53-HisTag, SEQ ID NO: 30) against anti-PD-1 antibody in a dilution series as mentioned above. FIG. 3B also shows a comparison of the reactivity of PD-L1 Fc monomer (SEQ ID NO:22) and PD-L1 p53 tetramer (SEQ ID NO:30).

2b. Comparison of PD-1 fusion proteins for quantification of anti-PD1 antibodies in human plasma

The recombinant proteins PD1-(GGGPS)2-lgG1-Fc (CH2, CH3 only) (SEQ ID.14), PD1-lgG1-Fc (SEQ ID.16) and PD1-p53 (SEQ ID NO:29) were tested for detection of anti-PD1 therapeutic mAbs used for cancer treatment as well as for determination of the anti-PD1 mAb drug concentration in the human plasma; in this case Pembrolizumab (Keytruda®) was used. ELISA plates were coated overnight with several PD-1 fusion proteins diluted in PBS, p.H. 7.4 as a coating buffer at a concentration of 50nM. The next day, plates were washed five times with IxELISA Wash buffer (BioRad, BUF031C). A blocking step was initiated by adding 200μί of 1 :4 BlockACE (BioRad, BUF029) water solution, prepared according to the instructions of the manufacturer. Plates were incubated for one hour at 37°C; after another washing step. Hundred microliters of each Pembrolizumab standard concentration, as well as an lgG1 negative control, were diluted in Assay buffer (1 :8 BlockACE water solution) and added to each well. Plates were incubated for one hour at 37°C and then washed five times with IxELISA wash buffer as previously described. Following this step, 100μί of

0.5u.g/mL whole mouse anti-human lgG4, Fc specific-conjugated with horseradish peroxidase antibody were added to each well (Thermoscientific, clone HP6023 cat. no. A-10654). Plates were incubated for thirty minutes at 37°C; washed five times in 1XELISA wash buffer and optical density was measured at 15, 30 and 60min; following addition of ABTS substrate (Roche, cat. no. 11 684302001).

Figure 4A show the use of PD-1 recombinant proteins to determine the concentrations of Pembrolizumab in human plasma. Pembrolizumab standards at six concentrations (ΙΟμς/πιΙ., ^g/mL, O.^g/mL, and 0.0001 ug/mL) were used to generate a standard curve for each of the recombinant PD1 proteins generated. The PD1 proteins tested are present at different oligomers states, 'monomer-like', dimer and tetramer. The data shows that PD1-p53 (tetramer) has higher avidity and lower EC50 value than PD1-Fc (dimer) and PD1-Fc (CH2, CH3) ('monomer'). In addition, using PD1-p53 provides an improved S/CO (signal to cut off noise) from concentrations ranging from 0.01-10μς/πιΙ (Fig. 4B). In this assay the cut-off value was determined by the mean of three negative plasma samples plus three times the standard deviation of the optical densities obtained for these samples.

2c. Reactivity of PD-L1 recombinant proteins

Different PD-L1 fusion proteins were tested for their reactivity against a specific anti-PD-L1 antibody. Recombinant versions of PD-L1 (PD-L1-p53 tetramer (SEQ ID NO:30) and PD-L1-Fc dimer (R&D systems)) were tested with fluorescently labeled anti-PD-L1 mAb. Briefly, ELISA plates (black) were coated with different PD-L1 proteins in equimolar concentrations, 20nM and detected directly with APC conjugated anti-PDL-1 antibody using a fluorescent reader. Figure 5 shows the comparison of PD-L1 Fc dimer (R&D sys) and PD-L1 p53 tetramer (SEQ ID NO:30) reactivity against anti-PD-L1 antibody in a serial dilution series (4 fold dilutions from 5μς/ιτιΙ to 0.001 μg/ml) and the signal to cut off to concentrations is depicted. The PD-L1 p53 tetramer showed higher binding reactivity than the PD-L1-Fc dimer. REFERENCES:

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