Expression of PD-l1 to improve gene therapy efficacy

Abstract

Disclosed herein are compositions comprising a vector encoding PD-L1 or fragment thereof and methods of use. In certain embodiments, the vector further encodes a peptide based therapeutic or binding agent. In certain embodiments, the compositions are used in methods of treating or preventing diseases or conditions that utilize peptide base therapeutics such as anti-viral, anti-HIV, anti-inflammatory, or anti-cancer antibodies.

Description

[0001] EXPRESSION OF PD-L1 TO IMPROVE GENE THERAPY EFFICACY

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 635,930 filed April 18, 2024. The entirety of this application is hereby incorporated by reference for all purposes.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under All 67724 and All 38860 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS AN XML FILE VIA THE OFFICE ELECTRONIC FILING SYSTEM

[0007] The Sequence Listing associated with this application is provided in XML format and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing is 24145PCT.xml. The XML file is 43,301 bytes, was created on April 17, 2025, and is being submitted electronically via the USPTO patent electronic filing system.

[0008] BACKGROUND

[0009] There are millions of humans living with HIV / AIDS. Combination antiretroviral therapy (ART) treatment regimens have successfully prolonged the lives of infected individuals. Stopping combination antiretroviral therapy (ART) leads to the HIV re-emergence. It is reported that HIV reservoirs residing in immune privileged areas are able to shield the virus from the immune system. See Churchill et al., Nat Rev Microbiol, 2016, 14:55-60. Thus, there is a need to identify improved methods of treating and preventing HIV.

[0010] Adeno-associated viral (AAV) vectors expressing therapeutic antibodies are reported to protected humanized mice from an HIV-1 challenge. However, a fraction of HIV-1 isolates remains partially or wholly resistant. Host immune responses can limit transgene expression. Clinical trials using AAV vectors to deliver HIV broadly neutralizing antibodies (bNAbs) by intramuscular administration resulted in poor expression with anti-drug antibodies (ADA) responses against the bNAb. Thus, there is a need to identify improved methods to minimize immune responses and facilitate expression of HIV broadly neutralizing antibodies (bNAbs).

[0011] Davis-Gardner et al. report a strategy for high antibody expression with low anti-drug antibodies using AAV9 vectors. Front Immunol, 2023, 14: 1105617

[0012] Gardner et al. report prophylaxis mediated by AAV-delivered HIV-1 broadly neutralizing antibodies. Molecular Therapy, 2019, 27(3):650.

[0013] Gardner et al. report AAV-expressed eCD4-Ig provides durable protection from multiple SHIV challenges Nature, 2015, 519(7541): 87-91.

[0014] Khatib et al. report beta-cell-targeted blockage of PD1 and CTLA4 pathways prevents development of autoimmune diabetes and acute allogeneic islets rejection. Gene Ther, 2015, 22(5), 430-438.

[0015] Li et al., report intra-articular delivery of AAV vectors encoding PD-L1 attenuates joint inflammation and tissue damage in a mouse model of rheumatoid arthritis. Front Immunol, 2023, 14: 1116084.

[0016] References cited herein are not an admission of prior art.

[0017] SUMMARY

[0018] Disclosed herein are compositions comprising a vector encoding PD-L1 or fragment thereof and a peptide based therapeutic or binding agent. In certain embodiments, the compositions are used in methods of treating or preventing disease or conditions that utilize peptide base therapeutics such as anti-viral, anti -HIV, anti-inflammatory, or anti-cancer antibodies.

[0019] In certain embodiments, this disclosure relates to compositions comprising a vector encoding PD-L1 constructs disclosed herein and a vector encoding a peptide based therapeutic or antibody, e g., viral binding agent or HIV binding agent. In certain embodiments, the vector is a single vector or a first vector encoding PD-L1 construct disclosed herein and a second vector encoding the peptide based therapeutic agent, e.g., viral binding agent or HIV binding agent.

[0020] In certain embodiments, the vector encoding a PD-L1 construct disclosed herein and the vector encoding the peptide based therapeutic, viral binding agent, or HIV binding agent is a recombinant adeno-associated virus (AAV). In certain embodiments, the recombinant adeno- associated virus (AAV) is a recombinant adeno-associated virus 9 (AAV9). In certain embodiments, this disclosure relates to compositions comprising a vector encoding PD-L1 constructs disclosed herein and a peptide based therapeutic agent, e.g., therapeutic antibody, viral binding agent, or an HIV binding agent. In certain embodiments, the compositions are used in methods of treating or preventing HIV or other viral infections, cancers or other immune related conditions, e.g., joint inflammation, tissue damage, rheumatoid arthritis.

[0021] In certain embodiments, this disclosure relates to compositions comprising a vector encoding PD-L1 constructs disclosed herein in operable combination with a promoter and a vector encoding an HIV binding agent in operable combination with a promoter. In certain embodiments, the vector is a single vector or a first vector encoding PD-L1 constructs disclosed herein and a second vector encoding the peptide based therapeutic agent, viral binding agent, or HIV binding agent.

[0022] In certain embodiments, it is contemplated that the promoter in operable combination with or associated with PD-L1 transgene has faster expression kinetics when in cells (cytosol or nucleus) than the promoter used in operable combination with or associated with the peptide based therapeutic, e.g., antibody.

[0023] In certain embodiments, the antibody or binding fragment contains an antibody light chain and / or an antibody heavy chain encoded by a single polypeptide connected by a peptide that generates polyproteins. In certain embodiments, the peptide that generates polyproteins has the amino acid sequence of DXEXNPGP (SEQ ID NO: 1), wherein X is individually and independently at each occurrence any amino acid. In certain embodiments, the peptide that generates polyproteins comprises GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 2) (P2A).

[0024] In certain embodiments, the vector encoding the peptide based therapeutic, viral binding agent or an HIV binding agent in operable combination with a promoter is a chicken -actin (CBA) promoter. In certain embodiments, a simian virus 40 (SV40) intron is downstream of the CBA promoter and a Woodchuck Hepatitis Virus (WHV) Posttranscriptional Regulatory Element (WPRE) is downstream of the encoded peptide based therapeutic, e.g., antibody heavy chain and antibody light chain. In certain embodiments, the vector encoding PD-L1 in operable combination with a promoter is a cytomegalovirus (CMV) promoter. In certain embodiments, a SV40 intron is downstream of the CMV promoter and a WPRE is downstream of the encoded PD-L1.

[0025] In certain embodiments, this disclosure relates to compositions comprising a single vector encoding PD-L1 construct disclosed herein and encoding an HIV binding agent. In certain embodiments, this disclosure relates to compositions comprising a single vector encoding a PD- L1 construct disclosed herein and encoding a peptide based therapeutic, e.g., viral binding agent / HIV binding agent, in operable combination with a bidirectional enhancer.

[0026] In certain embodiments, the PD-L1 constructs disclosed herein comprises a signal peptide, IgV (immunoglobulin variable) motif, IgC (immunoglobulin constant) motif, a transmembrane region and a cytoplasmic tail, wherein a segment of or all of the IgC motif is deleted. In certain embodiments, a segment or all of the cytoplasmic tail is deleted. In certain embodiments, the segment of the cytoplasmic tail deleted is the C-terminal DTSSK (SEQ ID NO: 3) peptide sequence.

[0027] In certain embodiments, a linker sequence is added in place of the deleted IgC motif or segment thereof. In certain embodiments, the linker sequence comprises G and S amino acids. In certain embodiments, the linker sequence is a poly GGGS (SEQ ID NO: 4) sequence. In certain embodiments, the linker sequence contains less than 10, 20, or 30 amino acids.

[0028] In certain embodiments, this disclosure relates to methods of preventing or treating a disease or condition, e.g., viral infection or HIV comprising administering an effective amount of a single vector encoding a PD-L1 construct disclosed herein and encoding a peptide based therapeutic agent, e.g., HIV binding agent in operable combination with a bidirectional enhancer as reported herein to a subject in need thereof.

[0029] In certain embodiments, this disclosure relates to methods or compositions wherein a lipid particle is used to contain, administer and / or deliver the therapeutic, gene, mRNA, or vector or nucleic acid encoding the same.

[0030] In certain embodiments, this disclosure relates to pharmaceutical compositions comprising constructs disclosed herein. In certain embodiments, this disclosure relates to the production of a medicament comprising constructs disclosed herein for use in treating or preventing a condition disclosed herein.

[0031] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0032] Figures 1 A-1C illustrate a contemplated mechanism for improved gene therapy in cells.

[0033] Figure 1 A illustrates an AAV vector encoding for an antibody that enters and transduces a muscle cell or other target cell. The AAV delivers its genetic cargo into the nucleus of the cell where it persists as a circular episome (2). The transduced cell then produces the transgene encoded by the episome, e.g., an antibody (or other peptide based therapeutic agent) (3).

[0034] Figure IB illustrates that host immune responses directed toward the transduced cells limit the efficacy of AAV gene therapy. The AAV vector enters and transduces the target cell (1). The target cell then signals through MHC / TCR interactions the activation of T cells (2). The activated T cell, such as a cytolytic CD8+ T cell, are directed to the transduced cell to kill it; thus, decreasing the amount of transgene (e.g., antibody) produced (3).

[0035] Figure 1C illustrates a contemplated method of targeting the immune checkpoint pathway to deactivate immune cells. Using two AAV vector, one encoding PD-L1 is co-deliver with the AAV vector encoding the antibody which enter and transduce the target cell (1). Both vectors deliver their genetic cargo to the nucleus, forming persistent episomes (2). Using a promoter with faster kinetics on the PD-L1 transgene than the antibody promoter, PD-L1 can be expressed on the cellular membrane forming an immunologic shield to PD-1 on the T cell (3). PD-1 signaling then inhibits T cell activation and keeping T cells from acting upon the transduced cell (4). The transduced cell persists and can continue to express the gene therapy transgene (i.e. antibody) (5). Using a single vector or two vectors encoding both PD-L1 and the specific binding agent / antibody is contemplated.

[0036] Figure 2. illustrates the construction of two AAV vectors used in experiments with nonhuman primates. Abbreviations: CBA Pro, chicken-beta actin promoter; Int, intron, VH - Variable heavy chain of antibody; rh-IgGl-LS CH, rhesus macaque IgGl constant heavy chain domains 1 through 3 with M428L and N434S amino acid substitutions in the IgGl Fc; P2A, selfcleaving 2A peptide from porcine teschovirus-1; rh-CL, rhesus macaque constant light chain; WPRE, woodchuck posttranscriptional response element; pA, polyadenylation signal sequence; CMV Pro, cytomegalovirus immediate-early promoter; rhesus macaque PD-L1 (rhPD-Ll). Note that the CMV promoter is used on the PD-L1 transgene as it has been shown to have faster kinetics of expression compared to the slower CBA promoter used for the antibody expression.

[0037] Figure 3 shows data indicating delivery of AAV-expressed PD-L1 and HIV antibodies results in consistent and persistent expression in nonhuman primates. Shown is data on concentrations of antibody 10-1074 (top) or antibody 3BNC117 (bottom) as measured by ELISA. Groups that only received the AAV vector encoding the antibody are on the left. Groups that were co-administered PD-L1 vectors are in the middle. Average of the two groups is on the right. Vector dose was 2.5E+12 vector genomes per kg of each vector. Six of six macaques that received 10- 1074 and PD-L1 vectors maintained expression of 10-1074 throughout the study while only four of six macaques that did not receive PD-L1 vectors had persistent 10-1074 expression. Five of six macaques that received 3BNC117 and PD-L1 vectors maintained expression of 3BNC117 throughout the study while only one of six macaques that did not receive PD-L1 vectors had persistent 3BNC117 expression.

[0038] Figure 4 shows data on anti-drug antibody (ADA) responses from rhesus macaques administered AAV9 vectors encoding HIV antibodies. (Top) Endpoint titers for ADA responses against 10-1074 from rhesus macaques expressing 10-1074 as in Figure 3 measured by anti- 10- 1074 Fab ELISA. (Bottom) Endpoint titers for ADA responses against 3BNC117 from rhesus macaques expressing 3BNC117 in Figure 3 as measured by anti-3BNC117 Fab ELISA. Note that there were fewer macaques with measurable ADA and lower endpoint titers in both groups that received co-admini strati on of AAV9 vectors encoding PD-L1.

[0039] Figure 5 shows data indicating a correlation of ADA and antibody expression. Correlations of ADA were plotted as endpoint titer measured by ELISA vs. area under the curve (AUC) of expressed antibodies described in Figure 2 (10-1074 or 3BNC117). Note the high degree of negative correlation as shown by Pearson r correlation coefficient meaning that higher ADA correlates with lower expression of an antibody.

[0040] Figure 6 shows data indicating protective efficacy of AAV-expressed antibodies. Kaplan- Meier curves indicate the protective efficacy of expressed 10-1074 (left) or 3BNC117 (right) from repeated, low-dose intrarectal (IR) SHIV-AD8 challenges. These data indicate that the expressed antibodies are functional and prevent infection similar to HIV transmission events in humans.

[0041] Figure 7A illustrates a single AAV expression cassette designed to express a shortened version of PD-L1 with a second transgene, e.g., dual promoter AAV expression cassette to express PD-L1 and a second transgene (e.g. antibody or other peptide based therapeutic). Note that in order to make both transgenes fit, the PD-L1 gene was shortened from its natural 870 base pair length to less than 740 base pairs.

[0042] Figure 7B shows a schematic of shortened PD-L1 variants (shPD-Llv#). Note that changes in the cytoplasmic tail or removal of the Ig-like C2-type domain were made to shorten PD-L1. Abbreviations: SP, signal peptide; TM, transmembrane domain; CT, cytoplasmic tail; sCT, shortened cytoplasmic tail; (G4S)3, linker consisting of four glycine amino acids and one serine amino acid in triplicate.

[0043] Figure 8 provides data from an in vitro demonstration of functional, shortened PD-L1 variants. Shortened PD-L1 variants (Fig. 7B) were assayed in an in vitro cellular based assay where HEK293T cells were transfected to express the indicated PD-L1 variant and a TCR activator. Growth-arrested Jurkat T cells (top) or live Jurkat T cells (bottom) were then added. Luciferase was produced in the Jurkat cells upon TCR activation by the expressed TCR activator on HEK293T cells. However, luciferase expression is diminished in those Jurkat cells where PD- 1 signals inhibit T cell activation when bound to a functional PD-L1 variant. A decrease of RLU signal in shortened PD-L1 variants indicate the feasibility of engineering shorter, functional versions of PD-L1. Abbreviations: rhPD-Ll, rhesus macaque PD-L1; huPD-Ll, human PD-L1, TCR, TCR activator only positive control; mock, mock transfection of empty DNA expression plasmid; shPD-Llv#, shortened PD-L1 variant.

[0044] Figure 9A illustrates experiments.

[0045] Figure 9B shows data on serum ADA endpoint titers determined by 3BNC117 ELISA (left) and correlation between bNAb serum level area under the curve and ADA endpoint titers at week 12 across all groups (right).

[0046] DETAILED DESCRIPTION

[0047] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims or as amended during prosecution.

[0048] Unless defined otherwise, 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 disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0049] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0050] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0051] An "embodiment" of this disclosure refers to an example and infers that the example is not necessarily limited to the example. Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

[0052] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

[0053] As used in this disclosure and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") have the meaning ascribed to them in U.S. Patent law in that they are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. The term “comprising” in reference to a protein having a peptide sequence refers to a peptide that may contain additional 5’ (5’ terminal end) or 3’ (3’ terminal end) nucleotides or N- or C-terminal amino acids, i.e., the term is intended to include the peptide sequence within a larger peptide. "Consisting essentially of' or "consists of' or the like, have the meaning ascribed to them in U.S. Patent law in that when applied to methods and compositions encompassed by the present disclosure refers to the idea of excluding certain prior art element(s) as an inventive feature of a claim, but which may contain additional composition components or method steps, etc., that do not materially affect the basic and novel character! stic(s) of the compositions or methods, compared to those of the corresponding compositions or methods disclosed herein.

[0054] The term “consisting of’ in reference to a peptide having an amino acid sequence refers to a peptide having the exact number of amino acids in the sequence and not more or having not more than a range of amino acids expressly specified in the claim. In certain embodiments, the disclosure contemplates that the “N-terminus of a peptide consists of an amino acid sequence,” which refers to the N-terminus of the peptide having the exact number of amino acids in the sequence and not more or having not more than a range of amino acids specified in the claim however the C-terminus may be connected to additional amino acids, e.g., as part of a larger peptide. Similarly, the disclosure contemplates that the “C-terminus of a peptide consists of an amino acid sequence,” which refers to the C-terminus of the peptide having the exact number of amino acids in the sequence and not more or having not more than a range of amino acids specified in the claim however the N-terminus may be connected to additional amino acids, e.g., as part of a larger peptide. In certain embodiments, the C-terminus may have 5, 10, 20, or 50 additional amino acids. In certain embodiments, the N-terminus may have 5, 10, 20, or 50 additional amino acids.

[0055] In certain contexts, an “antibody” refers to a protein-based molecule that is naturally produced by animals in response to the presence of a protein or other molecule or that is not recognized by the animal’s immune system to be a “self’ molecule, i.e., recognized by the animal to be a foreign molecule, i.e., an antigen to the antibody. The immune system of the animal will create an antibody to specifically bind the antigen, and thereby targeting the antigen for degradation or elimination, or any cell or organism attached to the antigen. It is well recognized by skilled artisans that the molecular structure of a natural antibody can be synthesized and altered by laboratory techniques. Recombinant engineering can be used to generate fully synthetic antibodies or fragments thereof providing control over variations of the amino acid sequences of the antibody. Thus, the term “antibody” is intended to include natural antibodies, monoclonal antibody, or non-naturally produced synthetic antibodies, such as specific binding single chain antibodies, bispecific antibodies, or fragments thereof. These antibodies may have chemical modifications. The term "monoclonal antibodies" refers to a collection of antibodies encoded by the same nucleic acid molecule that are optionally produced by a single hybridoma (or clone thereof) or other cell line, or by a transgenic mammal such that each monoclonal antibody will typically recognize the same antigen. The term "monoclonal" is not limited to any particular method for making the antibody, nor is the term limited to antibodies produced in a particular species, e.g., mouse, rat, etc.

[0056] From a structural standpoint, a human antibody is a combination of proteins: two heavy chain proteins and two light chain proteins. The heavy chains are longer than the light chains. The two heavy chains typically have the same amino acid sequence. Similarly, the two light chains typically have the same amino acid sequence. Often each of the heavy and light chains contain a variable segment that contains amino acid sequences which participate in binding to the antigen. The variable segments of the heavy chain do not have the same amino acid sequences as the light chains. The variable segments are often referred to as the antigen binding domains. The antigen and the variable regions of the antibody may physically interact with each other at specific smaller segments of an antigen often referred to as the "epitope." Epitopes usually consist of surface groupings of molecules, for example, amino acids or carbohydrates. The terms “variable region,” "antigen binding domain," and "antigen binding region" refer to that portion of the antibody molecule which contains the amino acid residues that interact with an antigen and confer on the antibody its specificity and affinity for the antigen. Small binding regions within the antigenbinding domain that typically interact with the epitope are also commonly referred to as the "complementarity-determining regions, or CDRs."

[0057] In certain embodiments, this disclosure contemplates fusion constructs comprising an antibody heavy chain (Fc domain) and known mutations. As used herein, a “mutation,” “mutant,” or the like of an antibody heavy chain sequence refers to the expression of a variant amino acid(s) within a heavy chain antibody defined by positions compared to base amino acids within the sequence segment, e.g., of UNIPROTKB / SWISS-PROT having accession number PO 1857.1.

[0058] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<A LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPP VLD SDGSFFLYSKLTVDKSRWQQGNVF SC S VMHEALHNHYTQKSL SL SPGK (SEQ ID NO: 5), wherein the N-terminal amino acid glutamic acid (E) is position 216. In certain embodiments, methionine (M) in bold and asparagine (N) in bold may be M428L and N434S amino acid substitutions in the IgGl .

[0059] The mutants may be constructed by building peptide sequences synthetically or, more typically, constructed using recombinant nucleic acid techniques, e.g., expression of the heavy chain in a cell from a template nucleic acid. Due to three codon translation of amino acids from nucleic acid, several three nucleotide codons may express the same amino acid variant. Sometimes the variant is due to a single nucleotide change, and sometimes the variant is due to more than one nucleotide change. Thus, reference to a “mutation,” “mutant,” or the like of a heavy chain antibody sequence are not necessarily limited to solely single nucleotide changes. In certain embodiments, the first or second heavy chain further has one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more of the following mutations G236A, S239D, A330L, I332E, S267E, L328F, P238D, H268F, S324T, S228P, G236R, L328R, L234A, L235A, M252Y, S254T, T256E, M428L, N434S, P329G, D265A, N297A, N297G, N297Q, F243L, R292P, Y300L, V305I, P396L, S298A, E333A, K334A, L234Y, L235Q, G236W, S239M, H268D, D270E, K326D, A330M, K334E, K326W, E333S, E345R, E430G, S440Y, L235E, N325S, wherein the mutation is in reference to positions in amino acid sequence (SEQ ID NO: 5) (segment of UNIPROTKB / SWISS-PROT: P01857.1), wherein the N-terminal glutamic acid (E) is position 216.

[0060] The CDRs are within the light and heavy chain variable segments of an antibody are sufficient for binding the epitope. Connecting the light and heavy chain variable segments (scFvs) together are often referred to as a single-chain variable fragments (scFvs). Sometimes only one antigen-binding domain is needed to bind the epitope, and the immune systems of some animals, e.g. camelids and sharks, are limited the production of single-chain antibodies which are typically resemble a single heavy chain. Thus, the term “antibody” is intended to include single-chain antibodies and antigen binding fragments. Other fish, such as lamprey, contain single-chain antibodies containing multiple leucine rich repeats.

[0061] A "chimeric antibody" is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules such that the entire molecule is not naturally occurring. Examples of chimeric antibodies include those having a variable region derived from a non-human antibody and a human immunoglobulin constant region, e.g. as illustrated above. The term is also intended to include antibodies having a variable region derived from one human antibody grafted to an immunoglobulin constant region of a predetermined sequences or the constant region from another human for which there are allotypic differences residing in the constant regions of any naturally occurring antibody having the variable regions, e.g., CDRs 1, 2, and 3 of the light and heavy chain. Human heavy chain genes exhibit structural polymorphism (allotypes) that are inherited as a haplotype. The serologically defined allotypes differ within and between population groups. See Jefferis et al. mAb, 1 (2009), pp. 332-338. In certain embodiments, the antibody, antibody heavy chain, antigen binding fragment, the light chain, or the heavy chain comprises a non-naturally occurring chimeric amino acid sequence such that there is at least one mutation that is not present in naturally occurring antibodies.

[0062] Smith et al. report a protocol for the production of antigen-specific chimeric human monoclonal antibodies (hmAbs) wherein antibody-secreting cells (ASCs) are isolated from whole blood collected after vaccination and sorted by flow cytometry into single cell plates. Nat Protoc. 2009;4(3):372-84. The antibody genes of the ASCs are then amplified by RT-PCR and nested PCR, cloned into expression vectors and transfected into a human cell line. Meijer et al. report methods for isolation of human antibody repertoires with preservation of the natural heavy and light chain pairing. J Mol Biol, 2006, 358(3):764-72. Wrammert et al. report using immunoglobulin variable regions isolated from sorted single ASCs to produce human monoclonal antibodies (mAbs) that bound with high affinity. Nature, 2008, 453(7195): 667-671.

[0063] Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, 1985, Science 229: 1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol. Methods 125: 191-202; and U.S. Pat. Nos. 6,311,415, 5,807,715, 4,816,567, and 4,816,397. Chimeric antibodies comprising one or more CDRs from a non-human species and framework regions from a human immunoglobulin molecule can be produced using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; International Publication No. WO 91 / 09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering 7:805; and Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969), and chain shuffling (U.S. Pat. No. 5,565,332).

[0064] The term "antibody fragment" refers to a peptide or polypeptide which comprises less than a complete, intact antibody. Complete antibodies comprise two functionally independent parts or fragments: an antigen binding fragment known as "Fab," and a carboxy terminal crystallizable fragment known as the "Fc" fragment. The Fab fragment includes the first constant domain from both the heavy and light chain (CHI and CL1) together with the variable regions from both the heavy and light chains that bind the specific antigen. Each of the heavy and light chain variable regions includes three complementarity determining regions (CDRs) and framework amino acid residues which separate the individual CDRs. The Fc region comprises the second and third heavy chain constant regions (CH2 and CH3) and is involved in effector functions such as complement activation and attack by phagocytic cells. In some antibodies, the Fc and Fab regions are separated by an antibody "hinge region," and depending on how the full length antibody is proteolytically cleaved, the hinge region may be associated with either the Fab or Fc fragment. For example, cleavage of an antibody with the protease papain results in the hinge region being associated with the resulting Fc fragment, while cleavage with the protease pepsin provides a fragment wherein the hinge is associated with both Fab fragments simultaneously. Because the two Fab fragments are in fact covalently linked following pepsin cleavage, the resulting fragment is termed the F(ab')2 fragment.

[0065] In certain embodiments, sequence "identity" refers to the number of exactly matching amino acids (expressed as a percentage) in a sequence alignment between two sequences of the alignment calculated using the number of identical positions divided by the greater of the shortest sequence or the number of equivalent positions excluding overhangs wherein internal gaps are counted as an equivalent position. In certain embodiments, any recitation of sequence identity expressed herein may be substituted for sequence similarity. Percent “similarity” is used to quantify the similarity between two sequences of the alignment. This method is identical to determining the identity except that certain amino acids do not have to be identical to have a match. Amino acids are classified as matches if they are among a group with similar properties according to the following amino acid groups: Aromatic - F Y W; hydrophobic-A V I L; Charged positive: R K H; Charged negative - D E; Polar - S T N Q.

[0066] In certain embodiments, this disclosure contemplates that a peptide based therapeutic is a therapeutic antibody, e.g., an antibody is selected from the group consisting of abagovomab, abciximab, abituzumab, abrezekimab, abrilumab, actoxumab, adalimumab, adecatumumab, aducanumab, afasevikumab, afelimomab, afutuzumab, alacizumab, alemtuzumab, alirocumab, altumomab, amatuximab, anatumomab, andecaliximab, anetumab, anifrolumab, anrukinzumab, apolizumab, aprutumab, arcitumomab, ascrinvacumab, aselizumab, atezolizumab, atinumab, atlizumab, atorolimumab, avelumab, azintuxizumab, bapineuzumab, basiliximab, bavituximab, bectumomab, begelomab, belantamab, belimumab, bemarituzumab, belimumab, bemarituzumab, benralizumab, berlimatoxumab, bersanlimab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bimagrumab, bimekizumab, birtamimab, bivatuzumab, bleselumab, blinatumomab, blontuvetmab, blosozumab, bococizumab, brazikumab, brentuximab, briakinumab, brodalumab, brolucizumab, brontictuzumab, burosumab, cabiralizumab, camidanlumab, camrelizumab, canakinumab, cantuzumab, caplacizumab, capromab, carlumab, carotuximab, catumaxomab, cedelizumab, cemiplimab, cergutuzumab, certolizumab, cetrelimab, cetuximab, cibisatamab, citatuzumab, cixutumumab, clazakizumab, clenoliximab, clivatuzumab, codrituzumab, cofetuzumab, coltuximab, conatumumab, concizumab, crenezumab crizanlizumab, crotedumab, cusatuzumab, dacetuzumab, daclizumab, dalotuzumab dapirolizumab, daratumumab, dectrekumab, demcizumab, denintuzumab, denosumab depatuxizumab, derlotuximab, detumomab, dezamizumab, dinutuximab, diridavumab domagrozumab, drozitumab, duligotuzumab, dupilumab, durvalumab, dusigitumab duvortuxizumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, eldelumab, elezanumab, elgemtumab, elotuzumab, emactuzumab, emapalumab, emibetuzumab, emicizumab, enapotamab, enavatuzumab, enfortumab, enoblituzumab, enokizumab, enoticumab, ensituximab, epitumomab, epratuzumab, eptinezumab, erenumab, erlizumab, ertumaxomab, etaracizumab, etigilimab, etrolizumab, evinacumab, evolocumab, exbivirumab, faralimomab, faricimab, farletuzumab, fasinumab, felvizumab, fezakinumab, fibatuzumab, ficlatuzumab, figitumumab, flanvotumab, fletikumab, flotetuzumab, fontolizumab, foralumab, foravirumab, fremanezumab, fresolimumab, frunevetmab, fulranumab, futuximab, galcanezumab, galiximab, gancotamab, ganitumab, gantenerumab, gatipotuzumab, gavilimomab, gedivumab, gemtuzumab, gevokizumab, gilvetmab, gimsilumab, girentuximab, glembatumumab, golimumab, gomiliximab, gosuranemab, guselkumab, ianalumab, ibalizumab, ibritumomab, icrucumab, idarucizumab, ifabotuzumab, igovomab, iladatuzumab, imalumab, imaprelimab, imciromab, imgatuzumab, inclacumab, indatuximab, indusatumab, inebilizumab, inflectra, infliximab, intetumumab, inolimomab, inotuzumab, ipilimumab, iratumumab, isatuximab, iscalimab, istiratumab, itolizumab, ixekizumab, keliximab, labetuzumab, lacnotuzumab, ladiratuzumab, lampalizumab, lanadelumab, landogrozumab, laprituximab, larcaviximab, lebrikizumab, lemalesomab, lendalizumab, lenvervimab, lenzilumab, lerdelimumab, leronlimab, lesofavumab, letolizumab, lexatumumab, libivirumab, lifastuzumab, ligelizumab, loncastuximab, losatuxizumab, lilotomab, lintuzumab, lirilumab, lodelcizumab, lokivetmab, lorvotuzumab, lucatumumab, lulizumab, lumiliximab, lumretuzumab, lupartumab, lutikizumab, mapatumumab, margetuximab, marstacimab, maslimomab, mavrilimumab, matuzumab, mepolizumab, metelimumab, milatuzumab, minretumomab, mirikizumab, mirvetuximab, mitumomab, modotuximab, mogamulizumab, monalizumab, morolimumab, mosunetuzumab, motavizumab, moxetumomab, nacolomab, namilumab, naptumomab, naratuximab, narnatumab, natalizumab, navicixizumab, navivumab, naxitamab, nebacumab, necitumumab, nemolizumab, nerelimomab, nesvacumab, netakimab, nimotuzumab, nirsevimab, nivolumab, nofetumomab, obiltoxaximab, obinutuzumab, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, oleclumab, olendalizumab, olokizumab, omalizumab, onartuzumab, ontuxizumab, onvatilimab, opicinumab, oportuzumab, oregovomab, orticumab, otelixizumab, otilimab, otlertuzumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, pamrevlumab, panitumumab, pankomab, panobacumab, parsatuzumab, pascolizumab, pasotuxizumab, pateclizumab, patritumab, pembrolizumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pinatuzumab, pintumomab, placulumab, plozalizumab, pogalizumab, polatuzumab, ponezumab, porgaviximab, prasinezumab, prezalizumab, priliximab, pritoxaximab, pritumumab, quilizumab, racotumomab, radretumab, rafivirumab, ralpancizumab, ramucirumab, ranevetmab, ranibizumab, raxibacumab, ravagalimab, ravulizumab, refanezumab, regavirumab, remtolumab, reslizumab, rilotumumab, rinucumab, risankizumab, rituximab, rivabazumab, robatumumab, roledumab, romilkimab, romosozumab, rontalizumab, rosmantuzumab, rovalpituzumab, rovelizumab, rozanolixizumab, ruplizumab, sacituzumab, samalizumab, samrotamab, sapelizumab, sarilumab, satralizumab, satumomab, secukinumab, selicrelumab, seribantumab, setoxaximab, setrusumab, sevirumab, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirtratumab, sirukumab, sofituzumab, solanezumab, solitomab, sonepcizumab, sontuzumab, spartalizumab, stamulumab, sulesomab, suptavumab, sutimlimab, suvizumab, suvratoxumab, tabalumab, tacatuzumab, tadocizumab, talacotuzumab, talizumab, tamtuvetmab, tanezumab, taplitumomab, tarextumab, tavolimab, tefibazumab, telimomab, telisotuzumab, tenatumomab, teneliximab, teplizumab, tepoditamab, teprotumumab, tesidolumab, tezepelumab, tibulizumab, tildrakizumab, tigatuzumab, timigutuzumab, timolumab, tiragolumab, tislelizumab, tisotumab, tocilizumab, toralizumab, tosatoxumab, tositumomab, tovetumab, tralokinumab, trastuzumab, tregalizumab, tremelimumab, trevogrumab, tucotuzumab, tuvirumab, ublituximab, urelumab, urtoxazumab, ustekinumab, utomilumab, vadastuximab, vanalimab, vandortuzumab, vantictumab, vanucizumab, vapaliximab, varisacumab, varlilumab, vatelizumab, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, vobarilizumab, volociximab, vonlerolizumab, vopratelimab, vorsetuzumab, votumumab, vunakizumab, xentuzumab, zalutumumab, zanolimumab, zenocutuzumab, ziralimumab, zolbetuximab, and zolimomab, or fragments thereof.

[0067] The terms “lipid particles” or “lipid nanoparticles” and the like refer to particles that have an average diameter of about or less than 1 mm or less than 500 nm in diameter that contain a phospholipid based exterior membrane. The phospholipid based exterior membranes typically contain a mixture of two or more of the following components a phospholipid, a sterol (cholesterol), glycol phospholipid, or ionizable lipid.

[0068] In addition to the nucleic acid sequences encoding the peptide(s) disclosed herein, recombinant expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Pat. Nos. 4,399,216; 4,634,665; and 5,179,017). For example, typically the selectable marker gene confers resistance to drugs (anti-microbial agents), such as G418, hygromycin, or methotrexate, on a host cell into which the vector has been introduced.

[0069] Standard molecular biology techniques can be used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the peptides or cells coated with the peptide from the culture medium. For example, the peptides or cells can be isolated by affinity chromatography.

[0070] In certain embodiments, this disclosure relates to nucleotide sequences or nucleic acids that encode the peptides or antibodies as disclosed herein, genetic constructs that include nucleotide sequences or nucleic acids and one or more elements. In certain embodiments, this disclosure relates to hosts or host cells (somatic cells) that contain such nucleotide sequences or nucleic acids, and / or that express (or are capable of expressing), the peptides disclosed herein.

[0071] In certain embodiments, this disclosure relates to methods for preparing peptides or antibodies containing the same or cells expressing the peptide or antibodies containing the same using constructs disclosed herein, which method comprises cultivating or maintaining a host cell under conditions such that said host cell produces or expresses the peptide(s) or antibody constructs thereof as disclosed herein.

[0072] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can comprise modified amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids such as homocysteine, ornithine, p- acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art.

[0073] The term "nucleic acid" refers to a polymer of nucleotides, or a polynucleotide, e.g., RNA, DNA, or a combination thereof. The term is used to designate a single molecule, or a collection of molecules. Nucleic acids may be single stranded or double stranded and may include coding regions and regions of various control elements.

[0074] A "heterologous" nucleic acid sequence or peptide sequence refers to a nucleic acid sequence or a peptide sequence that does not naturally occur, e.g., because the whole sequence contains a segment from other plants, bacteria, viruses, other organisms, or joinder of two sequences that occur the same organism but are j oined together in a manner that does not naturally occur in the same organism or any natural state.

[0075] The term "recombinant" when made in reference to a nucleic acid molecule refers to a nucleic acid molecule which is comprised of segments of nucleic acid joined together by means of molecular biological techniques provided that the entire nucleic acid sequence does not occurring in nature, i.e., there is at least one mutation in the overall sequence such that the entire sequence is not naturally occurring even though separately segments may occur in nature. The segments may be joined in an altered arrangement such that the entire nucleic acid sequence from start to finish does not naturally occur. The term "recombinant" when made in reference to a protein or a peptide refers to a protein molecule that is expressed using a recombinant nucleic acid molecule.

[0076] The terms "vector" or " expression vector " refer to a recombinant nucleic acid containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism or expression system, e.g., cellular or cell-free expression system. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.

[0077] In certain embodiments, this disclosure contemplates a vector encoding a peptide(s) or antibodies containing the same as disclosed herein in operable combination with a heterologous promoter or other transcription initiation element, translation initiation site, element that allows for translation initiation, translational enhancers, cis-acting nucleic acid DNA / RNA regions that promote internal initiation of protein synthesis. In certain embodiments, the promoter is a positive inducible promoter, negative inducible promoter, chemically inducible promoter, temperature inducible promoter, or light inducible promoter.

[0078] In certain embodiments, vectors comprise an internal ribosomal entry site(s) (IRES), internal ribosome entry site (IRES) to co-express heterologous gene products from a single promoter, e.g. multicistronic or bicistronic vectors contain IRES and / or self-cleaving (e.g. 2A) peptides to provide co-expression of multiple genes from a single RNA / DNA transcript. In certain embodiments, vectors are IRES-containing bicistronic vector for simultaneous expression of two proteins.

[0079] In certain embodiments, the disclosure relates to recombinant peptides and antibodies containing the same, comprising sequences disclosed herein or variants or fusions thereof wherein the interior amino acid sequence, the amino terminal end, or the carbon terminal end of the amino acid sequence are optionally attached to a heterologous amino acid sequence, label, or reporter molecule.

[0080] In certain embodiments, the disclosure relates to the recombinant vectors comprising a nucleic acid encoding a peptide or antibody containing the same as disclosed herein. In certain embodiments, the recombinant vector optionally comprises a mammalian, human, insect, viral, bacterial, bacterial plasmid, yeast associated origin of replication or gene such as a gene or retroviral gene or lentiviral LTR, TAR, RRE, PE, SLIP, CRS, and INS nucleotide segment or gene selected from tat, rev, nef, vif, vpr, vpu, and vpx or structural genes selected from gag, pol, and env. In certain embodiments, the recombinant vector optionally comprises a gene vector element (nucleic acid) such as a selectable marker region, lac operon, a CMV promoter, a hybrid chicken B-actin / CMV enhancer (CAG) promoter, tac promoter, T7 RNA polymerase promoter, SP6 RNA polymerase promoter, SV40 promoter, internal ribosome entry site (IRES) sequence, cis-acting woodchuck post regulatory element (WPRE), scaffold-attachment region (SAR), inverted terminal repeats (ITR), c-myc tag coding region, metal affinity tag coding region, streptavidin binding peptide tag coding region, polyHis tag coding region, HA tag coding region, MBP tag coding region, GST tag coding region, polyadenylation coding region, SV40 polyadenylation signal, SV40 origin of replication, Col El origin of replication, fl origin, pBR322 origin, or pUC origin, TEV protease recognition site, loxP sites, Cre recombinase coding region, or a multiple cloning site such as having 5, 6, or 7 or more restriction sites within a continuous segment of less than 50 or 60 nucleotides or having 3 or 4 or more restriction sites with a continuous segment of less than 20 or 30 nucleotides.

[0081] Nucleic acids and vectors encoding PD-L1 and a protein based therapeutic agent

[0082] Disclosed herein are compositions comprising a vector encoding PD-L1 and a therapeutic protein, e.g., an HIV antibody. In certain embodiments, this disclosure relates to compositions comprising a vector encoding PD-L1 in operable combination with a promoter and a vector encoding an HIV binding agent or other protein based therapeutic agent in operable combination with a promoter. In certain embodiments, the vector is a single vector or a first vector encoding PD-L1 and a second vector encoding the HIV binding agent or other protein based therapeutic agent.

[0083] In certain embodiments, the PD-L1 (CD274) sequences is a human sequence having the amino acid sequence

[0084] MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIV YWEMEDKNIIQF VHGEEDLK VQHS S YRQRARLLKDQL SLGNAALQITD VKLQD AGVYR CMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDH QVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHP PNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET (SEQ ID NO: 6) or variants thereof. In certain embodiments, variants have greater than 70%, 75%, 80%, 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, percent identity.

[0085] One can utilize computer programs to make stable amino acid substitutions of active variants and fragments can be identified with a high probability using computer modeling. Skilled artisans would know that certain conserved substitutions would be desirable one would avoid altering evolutionary conserved positions. See Saldano et al. Evolutionary Conserved Positions Define Protein Conformational Diversity, PLoS Comput Biol. 2016, 12(3):el004775. Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing biological activity may be found using computer programs in combination with publicly available databases well known in the art, for example, RaptorX, ESyPred3D, HHpred, Homology Modeling Professional for HyperChem, DNAStar, SPARKS-X, EVfold, Phyre, and Phyre2 software. See Kelley et al. which report the Phyre2 web portal for protein modelling, prediction and analysis. Nat Protoc, 2015, 10(6):845-58. See also Marks et al., Protein structure from sequence variation, Nat Biotechnol, 2012, 30(11): 1072-80; Mackenzie et al. Curr Opin Struct Biol, 2017, 44: 161-167; Mackenzie et al. Proc Natl Acad Sci U S A, 113(47):E7438-E7447 (2016) and Wei et al. Int J Mol Sci, 2016, 17(12), 2118.

[0086] Variants of a polypeptide with greater than identity of 90% can be easily produced by a skilled artisan, predicted for structural similarity using computer modeling, and tested for activity using procedures outlined in the examples of the specification.

[0087] In certain embodiments, this disclosure contemplates PD-L1 variants having 90% or greater identity. A skilled artisan would understand that one could produce a large number of operable PD-L1 variants with 90% identity that would be expected to have the desirable binding properties. PD genes are known and members shared significant homologies from one species to another. The sequences are not identical as illustrated by the differences between a human and a monkey sequences. For example, a comparison of SEQ ID NO: 6 (human) and SEQ ID NO: 7 (monkey) for the PD-L1 is shown below.

[0088] Human MRIFAVFI FMTYWHLLNAFTVTVPKDLYWEYGSNMTIECKFPVEKQLDLAALIVYWEME 60 MRIFAVFI F YWHLLNAFTVTVPKDLYWEYGSNMT+ECKFPVEKQLDL +LIVYWEME

[0089] Monkey MRIFAVFI FTIYWHLLNAFTVTVPKDLYVVEYGSNMTVECKFPVEKQLDLTSLIVYWEME 60

[0090] DKNI IQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMI SYGG 120 DKNI IQFVHGEEDLKVQHS+YRQRA+LLKDQLSLGNAAL+ITDVKLQDAGVYRCMI SYGG

[0091] DKNI IQFVHGEEDLKVQHSNYRQRAQLLKDQLSLGNAALRITDVKLQDAGVYRCMI SYGG 120

[0092] ADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTT 180 ADYKRITVKVNAP YNKI +QRI LWDPVT S EHELTCQAEGYPKAEVI WT S S DHQVLS GKTT ADYKRITVKVNAPYNKI SQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTT 180 TTNSKREEKLFNVTSTLRINTTTNEI FYCTFRRLDPEENHTAELVI PELPLAHPPNERTH 240

[0093] TTNSKREEKL NVTSTLRINTT NEI FYC FRRLDPEENHTAELVI PELPLA PPNERTH

[0094] TTNSKREEKLLNVTSTLRINTTANEI FYCI FRRLDPEENHTAELVI PELPLALPPNERTH 240

[0095] LVILGAI LLCLGVALTFI FRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET 290 ( SEQ ID NO : 6 )

[0096] LVILGAI L LGVALTFI F LRKGRMMD+KK GI + TNSKKQ DT LEET

[0097] LVILGAI FLLLGVALTFI FYLRKGRMMDMKKSGI RVTNSKKQRDTQLEET 290 ( SEQ ID NO : 7 )

[0098] Only 268 out of 290 amino acids (92%) are identical. Some are conserved substitutions

[0099] (plus sign). Some are not conserved substitutions. In certain embodiments, this disclosure contemplates variants having 92% or greater identity. In certain embodiments, this disclosure contemplates variants having 93% or greater identity. In certain embodiments, this disclosure contemplates variants having 94% or greater identity. In certain embodiments, this disclosure contemplates variants having 95% or greater identity. In certain embodiments, this disclosure contemplates variants having 96% or greater identity. In certain embodiments, this disclosure contemplates variants having 97% or greater identity. In certain embodiments, this disclosure contemplates variants having 98% or greater identity. In certain embodiments, this disclosure contemplates variants having 99% or greater identity.

[0100] In certain embodiments, this disclosure contemplates PD-L1 Domain 1 variants having 95% or greater identity. A comparison of SEQ ID NO: 8 (human) and SEQ ID NO: 9 (monkey) for the PD-L1 Domain 1 is shown below.

[0101] Human DI FTVTVPKDLYWEYGSNMTI ECKFPVEKQLDLAALIVYWEMEDKNI IQFVHGEEDLKVQH 60

[0102] FTVTVPKDLYWEYGSNMT+ECKFPVEKQLDL +LIVYWEMEDKNI IQFVHGEEDLKVQH

[0103] Monkey DI FTVTVPKDLYWEYGSNMTVECKFPVEKQLDLTSLIVYWEMEDKNI IQFVHGEEDLKVQH 60

[0104] S SYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMI SYGGADYKRITVKVNA 114 ( SEQ I D NO : 8 ) S+YRQRA+LLKDQLSLGNAAL+ITDVKLQDAGVYRCMI SYGGADYKRITVKVNA

[0105] SNYRQRAQLLKDQLSLGNAALRITDVKLQDAGVYRCMI SYGGADYKRITVKVNA 114 ( SEQ I D NO : 9 )

[0106] Only 108 out of 114 amino acids (95%) are identical. Some are conserved substitutions (plus sign). Some are not conserved substitutions. In certain embodiments, this disclosure contemplates DI variants having 95% or greater identity. In certain embodiments, this disclosure contemplates variants having 96% or greater identity. In certain embodiments, this disclosure contemplates variants having 97% or greater identity. In certain embodiments, this disclosure contemplates variants having 98% or greater identity. In certain embodiments, this disclosure contemplates variants having 99% or greater identity.

[0107] In certain embodiments, this disclosure contemplates PD-L1 Domain 2 variants having 95% or greater identity. A comparison of SEQ ID NO: 10 (human) and SEQ ID NO: 11 (monkey) for the PD-L1 Domain 2 is shown below.

[0108] Human D2 PYNKINQRI LWDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFN 60 PYNKI+QRI LWDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKL N

[0109] Monkey D2 PYNKI SQRI LWDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLLN 60

[0110] VTSTLRINTTTNEI FYCTFRRLDPEENHTAELV 93 ( SEQ I D NO : 10 ) VTSTLRINTT NEI FYC FRRLDPEENHTAELV VTSTLRINTTANEI FYCI FRRLDPEENHTAELV 93 ( SEQ I D NO : 11 )

[0111] Only 89 out of 93 amino acids (95%) are identical. Some are conserved substitutions (plus sign). Some are not conserved substitutions. In certain embodiments, this disclosure contemplates DI variants having 95% or greater identity. In certain embodiments, this disclosure contemplates variants having 96% or greater identity. In certain embodiments, this disclosure contemplates variants having 97% or greater identity. In certain embodiments, this disclosure contemplates variants having 98% or greater identity. In certain embodiments, this disclosure contemplates variants having 99% or greater identity.

[0112] In certain embodiments, this disclosure contemplates constructs that contain a signal peptide (SP) having MRIFAVFIFMTYWHLLNA (SEQ ID NO: 12) or variants thereof. In certain embodiments, variants have greater than 70%, 75%, 80%, 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, percent identity.

[0113] In certain embodiments, this disclosure contemplates constructs that contain a PD-L1 domain 1 and a transmembrane linker (Dl-TM). In certain embodiments, the linker has GGS, GGSGGS (SEQ ID NO: 13), GGSGGSGGS (SEQ ID NO: 14), GGSGGSGGSGGS (SEQ ID NO: 15), GGSGGSGGSGGSGGS (SEQ ID NO: 16), GGSGGSGGSGGSGGSGGS (SEQ ID NO: 17), GGSGGSGGSGGSGGSGGSGGS (SEQ ID NO: 18), GGSGGSGGSGGSGGSGGSGGSGGS (SEQ ID NO: 19), GGSGGSGGSGGSGGSGGSGGSGGSGGS (SEQ ID NO: 20), or GGSGGSGGSGGSGGSGGSGGSGGSGGSGGS (SEQ ID NO: 21) or variants. In certain embodiments, variants have greater than 70%, 75%, 80%, 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, percent identity.

[0114] In certain embodiments the linker has GGGGS (SEQ ID NO: 22), GGGGSGGGGS (SEQ ID NO: 23), GGGGSGGGGSGGGGS (SEQ ID NO: 24), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 25), or GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 26) or variants. In certain embodiments, variants have greater than 70%, 75%, 80%, 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, percent identity.

[0115] In certain embodiments the linker has SGSETPGTSESATPES (SEQ ID NO: 27, XTEN).

[0116] In certain embodiments the linker has EAAAK (SEQ ID NO: 28), EAAAKEAAAK (SEQ ID NO: 29), or EAAAKEAAAKEAAAK (SEQ ID NO: 30) or variants. In certain embodiments, variants have greater than 70%, 75%, 80%, 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, percent identity.

[0117] In certain embodiments, the linker has AEAAAKEAAAKA (SEQ ID NO: 31) or variants. In certain embodiments, variants have greater than 70%, 75%, 80%, 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, percent identity.

[0118] In certain embodiments, the linker has AEAAAKEAAAKEAAAKEAAAKALEAEAAA KEAAAKEAAAKEAAAKA(SEQ ID NO: 32) or variants. In certain embodiments, variants have greater than 70%, 75%, 80%, 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, percent identity.

[0119] In certain embodiments, constructs have a pre-transmembrane domain (PT). In certain embodiments, the pre-transmembrane domain has IPELPLAHPPNER (SEQ ID NO: 33) or variants. In certain embodiments, variants have greater than 70%, 75%, 80%, 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, percent identity.

[0120] In certain embodiments, constructs have a transmembrane domain (TM). In certain embodiments, the transmembrane domain (TM) has THLVILGAILLCLGVALTFIF (SEQ ID NO: 34) or variants. In certain embodiments, variants have greater than 70%, 75%, 80%, 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, percent identity.

[0121] In certain embodiments, constructs have a cytoplasmic tail (CT). In certain embodiments, the cytoplasmic tail has RLRKGRMMDVKKCGIQDTNSKKQSDTHLEET (SEQ ID NO: 35) or variants. In certain embodiments, variants have greater than 70%, 75%, 80%, 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, percent identity.

[0122] In certain embodiments, constructs have engineered shortened versions of PD-L1 and contain a signal peptide (from PD-L1 or other protein), the PD-L1 DI domain, and a transmembrane domain (from PD-L1 or other protein). In certain embodiments, constructs further optionally comprise a PD-L1 D2 domain, a linking group, phosphotyrosine-binding domain (PTB) or C-terminal domain (CTD) (from PD-L1 or other protein)

[0123] In certain embodiments, constructs have engineered shortened versions of PD-L1 is

[0124] FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEE DLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKV NA (SEQ ID NO: 8) or variants. In certain embodiments, variants have greater than 70%, 75%, 80%, 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, percent identity.

[0125] In certain embodiments, constructs have engineered PD-L1 human domain sequences such as

[0126] MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIV YWEMEDKNIIQF VHGEEDLKVQHS S YRQRARLLKDQL SLGNAALQITD VKLQD AGVYR CMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDH QVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHP PNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQ (SEQ ID NO: 36) or variants. In certain embodiments, variants have greater than 70%, 75%, 80%, 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, percent identity.

[0127] In certain embodiments, constructs have engineered PD-L1 human domain sequences such as

[0128] MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIV YWEMEDKNIIQF VHGEEDLKVQHS S YRQRARLLKDQL SLGNAALQITD VKLQD AGVYR CMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDH QVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHP PNERTHLVILGAILLCLGVALTFIF (SEQ ID NO: 37) or variants. In certain embodiments, variants have greater than 70%, 75%, 80%, 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, percent identity. In certain embodiments, constructs have engineered PD-L1 human domain sequences such as

[0129] MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIV YWEMEDKNIIQFVHGEEDLKVQHSS YRQRARLLKDQL SLGNAALQITD VKLQD AGVYR CMISYGGADYKRITVKVNAGGGGSGGGGSGGGGSIPELPLAHPPNERTHLVILGAILLCL GVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET (SEQ ID NO: 38) or variants. In certain embodiments, variants have greater than 70%, 75%, 80%, 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, percent identity.

[0130] In certain embodiments, constructs have engineered PD-L1 human domain sequences such as

[0131] MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIV YWEMEDKNIIQF VHGEEDLK VQHS S YRQRARLLKDQL SLGNAALQITD VKLQD AGVYR CMISYGGADYKRITVKVNAGGGGSGGGGSGGGGSIPELPLAHPPNERTHLVILGAILLCL GVALTFIFRLRKGRMMDVKKCGIQ (SEQ ID NO: 39) or variants. In certain embodiments, variants have greater than 70%, 75%, 80%, 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, percent identity.

[0132] In certain embodiments, constructs have engineered PD-L1 human domain sequences such as

[0133] MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIV YWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYR CMISYGGADYKRITVKVNAGGGGSGGGGSGGGGSIPELPLAHPPNERTHLVILGAILLCL GVALTFIF (SEQ ID NO: 40) or variants. In certain embodiments, variants have greater than 70%, 75%, 80%, 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, percent identity.

[0134] In certain embodiments, constructs have engineered PD-L1 domain sequences such as

[0135] MRIFAVFIFTIYWHLLNAFTVTVPKDLYVVEYGSNMTVECKFPVEKQLDLTSLIVY WEMEDKNIIQFVHGEEDLKVQHSNYRQRAQLLKDQLSLGNAALRITDVKLQDAGVYRC MISYGGADYKRITVKVNAPYNKISQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQV LSGKTTTTNSKREEKLLNVTSTLRINTTANEIFYCIFRRLDPEENHTAELVIPELPLALPPNE RTHLVILGAIFLLLGVALTFIFYLRKGRMMDMKKSGIR (SEQ ID NO: 41) or variants. In certain embodiments, variants have greater than 70%, 75%, 80%, 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, percent identity. In certain embodiments, constructs have engineered PD-L1 domain sequences such as MRIFAVFIFTIYWHLLNAFTVTVPKDLYVVEYGSNMTVECKFPVEKQLDLTSLIVY WEMEDKNIIQFVHGEEDLKVQHSNYRQRAQLLKDQLSLGNAALRITDVKLQDAGVYRC MISYGGADYKRITVKVNAPYNKISQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQV LSGKTTTTNSKREEKLLNVTSTLRINTTANEIFYCIFRRLDPEENHTAELVIPELPLALPPNE RTHLVILGAIFLLLGVALTFIFYL (SEQ ID NO: 42) or variants. In certain embodiments, variants have greater than 70%, 75%, 80%, 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, percent identity.

[0136] In certain embodiments, constructs have engineered PD-L1 domain sequences such as MRIFAVFIFTIYWHLLNAFTVTVPKDLYVVEYGSNMTVECKFPVEKQLDLTSLIVY WEMEDKNIIQFVHGEEDLKVQHSNYRQRAQLLKDQLSLGNAALRITDVKLQDAGVYRC MISYGGADYKRITVKVNAGGGGSGGGGSGGGGSIPELPLALPPNERTHLVILGAIFLLLG VALTFIFYLRKGRMMDMKKSGIRVTNSKKQRDTQLEET (SEQ ID NO: 43) or variants. In certain embodiments, variants have greater than 70%, 75%, 80%, 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, percent identity.

[0137] In certain embodiments, constructs have engineered PD-L1 domain sequences such as

[0138] MRIFAVFIFTIYWHLLNAFTVTVPKDLYVVEYGSNMTVECKFPVEKQLDLTSLIVY WEMEDKNIIQFVHGEEDLKVQHSNYRQRAQLLKDQLSLGNAALRITDVKLQDAGVYRC MISYGGADYKRITVKVNAGGGGSGGGGSGGGGSIPELPLALPPNERTHLVILGAIFLLLG VALTFIFYLRKGRMMDMKKSGIR (SEQ ID NO: 44) or variants. In certain embodiments, variants have greater than 70%, 75%, 80%, 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, percent identity.

[0139] In certain embodiments, constructs have engineered PD-L1 domain sequences such as MRIFAVFIFTIYWHLLNAFTVTVPKDLYVVEYGSNMTVECKFPVEKQLDLTSLIVY WEMEDKNIIQFVHGEEDLKVQHSNYRQRAQLLKDQLSLGNAALRITDVKLQDAGVYRC MISYGGADYKRITVKVNAGGGGSGGGGSGGGGSIPELPLALPPNERTHLVILGAIFLLLG VALTFIFYL (SEQ ID NO: 45) or variants. In certain embodiments, variants have greater than 70%, 75%, 80%, 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, percent identity.

[0140] In certain embodiments, the vector encoding PD-L1 or fragment thereof and the vector encoding an HIV binding agent is a recombinant Adeno-associated vims (AAV). In certain embodiments, the recombinant Adeno-associated virus (AAV) is a recombinant Adeno-associated virus 9 (AAV9). In certain embodiments, the encoded HIV binding agent is an antibody or binding fragment thereof.

[0141] In certain embodiments, the antibody or binding fragment contains an antibody light chain and antibody heavy chain encoded by a single polypeptide connected by a peptide that generates polyproteins. In certain embodiments, the peptide that generates polyproteins has the amino acid sequence of DXEXNPGP (SEQ ID NO: 1), wherein X is individually and independently at each occurrence any amino acid. In certain embodiments, the peptide that generates polyproteins is (P2A) GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 2).

[0142] In certain embodiments, the vector encoding an HIV binding agent in operable combination with a promoter is a CBA promoter. In certain embodiments, a SV40 intron is downstream of the CBA promoter and a WPRE is downstream of the encoded antibody heavy chain and antibody light chain. In certain embodiments, the vector encoding PD-L1 in operable combination with a promoter is a CMV promoter. In certain embodiments, a SV40 intron is downstream of the CMV promoter and a WPRE is downstream of the encoded PD-L1.

[0143] In certain embodiments, this disclosure relates to compositions comprising a single vector encoding PD-L1 or fragment and encoding an HIV binding agent or fragment. In certain embodiments, this disclosure relates to compositions comprising a single vector encoding PD-L1 or fragment and encoding an HIV binding agent for fragment in operable combination with a bidirectional enhancer. In certain embodiments, the single vector encoding PD-L1 or fragment and encoding an HIV binding agent or fragment is a recombinant adeno-associated virus (AAV).

[0144] In certain embodiments, the PD-L1 protein comprises a signal peptide, IgV motif, IgC motif, a transmembrane region and a cytoplasmic tail, and wherein a segment of or all of the IgC motif is deleted. In certain embodiments, a segment or all of the cytoplasmic tail is deleted. In certain embodiments, the segment of the cytoplasmic tail deleted is the C-terminal DTSSK (SEQ ID NO: 3) peptide sequence.

[0145] In certain embodiments, a linker sequence is added in place of the deleted IgC motif or segment thereof. In certain embodiments, the linker sequence comprises G and S amino acids. In certain embodiments, the linker sequence is a poly GGGS (SEQ ID NO: 4) sequence. In certain embodiments, the linker sequence contains less than 20 or 30 amino acids. Methods of managing peptide therapies

[0146] In certain embodiments, this disclosure relates to methods of preventing or treating a disease or condition comprising administering an effective amount of a vector encoding PD-L1 constructs disclosed herein and encoding a peptide based therapeutic agent to a subject in need thereof. In certain embodiments, the subject is diagnosed with a viral infection, e.g., HIV infections, cancer, or an immune inflammatory related condition.

[0147] As used herein, "subject" refers to any animal, preferably a human patient, livestock, or domestic pet.

[0148] The term “effective amount” refers to that amount of an agent or pharmaceutical composition described herein that is sufficient to effect the intended application including, but not limited to, prevention of microbial infection or disease treatment. In relation to a combination therapy, an “effective amount” indicates the combination of agent results in synergistic or additive effect when compared to the agents individually. The therapeutically effective amount can vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The specific dose will vary depending on, for example, the particular compounds chosen, the dosing regimen to be followed, whether it is administered in combination with other agents, timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.

[0149] As used herein, the terms "prevent" and "preventing" include the prevention of the recurrence, spread or onset. It is not intended that the present disclosure be limited to complete prevention. In some embodiments, the onset is delayed, or the severity of the disease is reduced.

[0150] As used herein, the terms "treat" and "treating" are not limited to the case where the subject (e.g., patient) is cured and the disease is eradicated. Rather, embodiments, of the present disclosure also contemplate treatment that merely reduces symptoms, and / or delays disease progression.

[0151] As used herein, the term "combination with" when used to describe administration of an agent with an additional treatment means such that the agent may be administered prior to, together with, or after the additional treatment, or a combination thereof, such that multiple agents are pharmacologically available at some overlapping time, e.g. considering the pharmacokinetic properties, e.g., half-life of each agent. The terms “viral infection” or “viral disease” or “viral infectious disease” or “virus infection” as used interchangeably herein refers, in the usual and customary sense, to the presence of a virus (e.g., virus) within a subject. In embodiments, a viral infection refers to the presence of a virus (e.g., virus) within a subject that is capable of replicating and / or generating virus particles. In embodiments, the viral infection refers to the presence of a virus (e.g., virus) within a subject that is capable of infecting a second subject. A viral infection can be present in any body issue and the subject may present symptoms such as fever, red eyes, joint pain, headache, and a maculopapular rash, or the subject may be asymptomatic. Diagnosis of a viral infection may be determined by testing bodily fluids (e.g., blood, urine, or saliva) for the presence of the virus's RNA / DNA or for antibodies. In embodiments, the virus may be present within a subject but may be latent.

[0152] In certain embodiments, this disclosure relates to methods of preventing or treating a disease or condition comprising administering an effective amount of a vector encoding PD-L1 construct disclosed herein in operable combination with a promoter and a vector encoding a peptide based therapeutic agent, e.g., an HIV binding agent or other viral binding agent in operable combination with a promoter other translation initiation site to a subject in need thereof.

[0153] In certain embodiments, the vectors disclosed herein are based on an adenovirus / ad enoassociated virus; however, other constructs of recombinant virus vectors expressing PD-L1 and peptide based therapeutic agents are contemplated including vectors based on poxviruses, iridoviruses, Simian Virus 40 (S V40), Epstein-Barr virus, herpesvirus, IC virus, bacteriophage T7, bacteriophage, T3 and bacteriophage SP6.

[0154] In certain embodiments, the vector encoding PD-L1 constructs disclosed herein and / or the vector encoding the peptide based therapeutic, viral binding agent, or HIV binding agent is an adenovirus, adeno-associated virus (AAV), vaccinia virus, poxviruses virus, herpes simplex virus, measles virus, vesicular stomatitis virus, poliovirus, alphaviruses, lentivirus, enterovirus, poliovirus, paramyxoviruses, and / or reovirus.

[0155] It will be understood by those skilled in the art that because recombinant virus vectors of this disclosure are made by starting with a selected virus (referred to herein as the base or originating virus) and then making alterations to the genome thereof, the majority of the structure (i.e., nucleic acid molecules, proteins, etc.) of the recombinant virus vector will come from the base virus, i.e., final recombinant virus vector will comprise the genome of the base virus, albeit with the necessary alterations made thereto. Consequently, the final recombinant virus vector can be referred to with reference to the base virus. For example, if a recombinant virus vector of this disclosure is constructed starting with an adeno-associated virus, the majority of the nucleic acid molecules and proteins in the recombinant virus vector will come from an adenovirus virus and thus the final recombinant virus vector can be referred to, for example, as a recombinant adeno- associated viral vector. In certain embodiments, it is contemplated that sequences disclosed herein can be integrated into other recombinant viral vectors, e.g., recombinant poxvirus vector, a recombinant vaccinia virus vector, a recombinant chordopoxvirus vector, a recombinant iridovirus vector, a recombinant mimiviral vector, a recombinant SV40 virus vector, a recombinant Epstein- Barr virus vector, and a recombinant herpes virus vector.

[0156] In certain embodiments, this disclosure relates to methods of preventing or treating a viral infection, e.g., HIV, cancer, or immune related condition comprising administering an effective amount of a vector encoding a PD-L1 construct disclosed herein in operable combination with a promoter and a vector encoding an HIV binding agent or other peptide based viral agent in operable combination with a promoter.

[0157] In certain embodiments, this disclosure relates to methods of preventing or treating a viral infection, HIV, cancer, or immune related condition comprising administering an effective amount of a vector encoding PD-L1 construct disclosed herein in operable combination with a promoter and a vector encoding an HIV binding agent or other peptide based viral agent, cancer agent, or immunosuppressive agent in operable combination with a promoter to a subject in need thereof.

[0158] In certain embodiments, this disclosure relates to methods of preventing or treating HIV comprising administering an effective amount of a vector encoding PD-L1 construct disclosed herein in operable combination with a promoter and a vector encoding an HIV binding agent in operable combination with a promoter to a subject in need thereof

[0159] In certain embodiments, this disclosure relates to methods of preventing or treating HIV comprising administering an effective amount of a single vector encoding PD-L1 and encoding an HIV binding agent in operable combination with a bidirectional enhancer as reported herein to a subject in need thereof.

[0160] In certain embodiments, this disclosure relates to methods or compositions as disclosed herein wherein use of a lipid particle / nanoparticle is used to contain, administer and / or deliver the therapeutic, gene, mRNA, DNA, or vector or nucleic acid encoding the same. In certain embodiments, this disclosure relates to methods of preventing or treating HIV comprising administering an effective amount of a vector encoding a PD-L1 construct disclosed herein and an HIV binding agent or HIV drug to a subj ect in need thereof. In certain embodiments, the HIV binding agent is a peptide based binding agent or a small molecule binding agent or drug.

[0161] In certain embodiments, this disclosure relates to methods of preventing or treating HIV comprising administering an effective amount of 1) a vector encoding a PD-L1 construct or PD- L1 peptide disclosed herein and encoding an HIV binding agent, in combination with administering 2) HIV drug(s) to a subject in need thereof. In certain embodiments, the HIV binding agent is a peptide based binding agent or a small molecule binding agent or drug. In certain embodiments, the drug is an HIV drug or combination of drugs HIV antiretroviral therapies (ART).

[0162] In certain embodiments, this disclosure relates to methods of preventing or treating HIV comprising administering an effective amount of 1) a vector encoding a PD-L1 construct or PD- Lf peptide disclosed herein and 2) an HIV binding agent and / or HIV drug(s) to a subject in need thereof. In certain embodiments, the HIV binding agent is a peptide based binding agent or a small molecule binding agent or drug. In certain embodiments, the drug is an HIV drug or combination of drugs.

[0163] In certain embodiments, the constructs disclosed herein are administered to a human patient in need thereof are also administered in combination with a multi-drug anti-retroviral therapy (ART). In certain embodiments, the multi-drug anti-retroviral therapy comprises a nucleoside reverse transcriptase inhibitor (NRTI), abacavir, emtricitabine, lamivudine, tenofovir disoproxil fumarate, zidovudine, non-nucleoside reverse transcriptase inhibitor (NNRTI), doravirine, efavirenz, etravirine, nevirapine, rilpivirine, protease inhibitor (PI), atazanavir, darunavir, fosamprenavir, ritonavir, tipranavir, fusion inhibitor, enfuvirtide, CCR5 antagonist, maraviroc, capsid Inhibitor, lenacapavir, attachment inhibitor, fostemsavir, post-attachment inhibitor, ibalizumab, ibalizumab, integrase strand transfer inhibitor (INSTI), cabotegravir, dolutegravir, raltegravir, or combinations thereof.

[0164] In certain embodiments, the combination is abacavir and lamivudine; or abacavir, dolutegravir, and lamivudine; or abacavir, lamivudine, and zidovudine; or atazanavir and cobicistat; or bictegravir, emtricitabine, and tenofovir alafenamide; or cabotegravir and rilpivirine; or darunavir and cobicistat; or darunavir, cobicistat, emtricitabine, and tenofovir alafenamide; or dolutegravir and lamivudine; or dolutegravir and rilpivirine; or doravirine, lamivudine, and tenofovir disoproxil fumarate; or efavirenz, emtricitabine, and tenofovir disoproxil fumarate; or efavirenz, lamivudine, and tenofovir disoproxil fumarate; or elvitegravir, cobicistat, emtricitabine, and tenofovir alafenamide; or elvitegravir, cobicistat, emtricitabine, and tenofovir disoproxil fumarate; or emtricitabine, rilpivirine, and tenofovir alafenamide; or emtricitabine, rilpivirine, and tenofovir disoproxil fumarate; or emtricitabine and tenofovir alafenamide; or emtricitabine and tenofovir disoproxil fumarate; or lamivudine and tenofovir disoproxil fumarate; or lamivudine and zidovudine; or lopinavir and ritonavir.

[0165] In certain embodiments, this disclosure relates to methods of preventing or treating cancer or immune related condition comprising administering an effective amount of a vector encoding PD-L1 construct disclosed herein in operable combination with a promoter and a vector encoding a peptide based anti-cancer agent or immune suppressive agent in operable combination with a promoter optionally in combination with other anti-cancer agents or anti-inflammatory agents.

[0166] In certain embodiments, the peptide based anti-cancer agent is an anti-PD-1, anti-CTLA4 antibody or combinations thereof, such as an anti-CTLA4 (e.g., ipilimumab, tremelimumab) and anti-PDl (e g., nivolumab, pembrolizumab, cemiplimab).

[0167] "Cancer" refers any of various cellular diseases with malignant neoplasms characterized by the proliferation of cells. It is not intended that the diseased cells must actually invade surrounding tissue and metastasize to new body sites. Cancer can involve any tissue of the body and have many different forms in each body area. Within the context of certain embodiments, whether "cancer is reduced" may be identified by a variety of diagnostic manners known to one skill in the art including, but not limited to, observation the reduction in size or number of tumor masses or if an increase of apoptosis of cancer cells observed, e.g., if more than a 5 % increase in apoptosis of cancer cells is observed for a sample compound compared to a control without the compound. It may also be identified by a change in relevant biomarker or gene expression profile, such as PSA for prostate cancer, HER2 for breast cancer, or other.

[0168] The cancer to be treated in the context of the present disclosure may be any type of cancer or tumor. These tumors or cancer include, and are not limited to, tumors of the hematopoietic and lymphoid tissues or hematopoietic and lymphoid malignancies, tumors that affect the blood, bone marrow, lymph, and lymphatic system. Hematological malignancies may derive from either of the two major blood cell lineages: myeloid and lymphoid cell lines. The myeloid cell line normally produces granulocytes, erythrocytes, thrombocytes, macrophages and mast cells; the lymphoid cell line produces B, T, NK and plasma cells. Lymphomas, lymphocytic leukemias, and myeloma are from the lymphoid line, while acute and chronic myelogenous leukemia, myelodysplastic syndromes and myeloproliferative diseases are myeloid in origin.

[0169] Also contemplated are malignancies located in the colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, hypophysis, testicles, ovaries, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thorax and urinary apparatus and, more particularly, childhood acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, adrenocortical carcinoma, adult (primary) hepatocellular cancer, adult (primary) liver cancer, adult acute lymphocytic leukemia, adult acute myeloid leukemia, adult Hodgkin's disease, adult Hodgkin's lymphoma, adult lymphocytic leukemia, adult non-Hodgkin's lymphoma, adult primary liver cancer, adult soft tissue sarcoma, AIDS-related lymphoma, AIDS-related malignant tumors, anal cancer, astrocytoma, cancer of the biliary tract, cancer of the bladder, bone cancer, brain stem glioma, brain tumors, breast cancer, cancer of the renal pelvis and ureter, primary central nervous system lymphoma, central nervous system lymphoma, cerebellar astrocytoma, brain astrocytoma, cancer of the cervix, childhood (primary) hepatocellular cancer, childhood (primary) liver cancer, childhood acute lymphoblastic leukemia, childhood acute myeloid leukemia, childhood brain stem glioma, childhood cerebellar astrocytoma, childhood brain astrocytoma, childhood extracranial germ cell tumors, childhood Hodgkin's disease, childhood Hodgkin's lymphoma, childhood visual pathway and hypothalamic glioma, childhood lymphoblastic leukemia, childhood medulloblastoma, childhood non-Hodgkin's lymphoma, childhood supratentorial primitive neuroectodermal and pineal tumors, childhood primary liver cancer, childhood rhabdomyosarcoma, childhood soft tissue sarcoma, childhood visual pathway and hypothalamic glioma, chronic lymphocytic leukemia, chronic myeloid leukemia, cancer of the colon, cutaneous T-cell lymphoma, endocrine pancreatic islet cells carcinoma, endometrial cancer, ependymoma, epithelial cancer, cancer of the esophagus, Ewing's sarcoma and related tumors, cancer of the exocrine pancreas, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic biliary tract cancer, cancer of the eye, breast cancer in women, Gaucher's disease, cancer of the gallbladder, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal tumors, germ cell tumors, gestational trophoblastic tumor, tricoleucemia, head and neck cancer, hepatocellular cancer, Hodgkin's disease, Hodgkin's lymphoma, hypergammaglobulinemia, hypopharyngeal cancer, intestinal cancers, intraocular melanoma, islet cell carcinoma, islet cell pancreatic cancer, Kaposi's sarcoma, cancer of kidney, cancer of the larynx, cancer of the lip and mouth, cancer of the liver, cancer of the lung, lymphoproliferative disorders, macroglobulinemia, breast cancer in men, malignant mesothelioma, malignant thymoma, medulloblastoma, melanoma, mesothelioma, occult primary metastatic squamous neck cancer, primary metastatic squamous neck cancer, metastatic squamous neck cancer, multiple myeloma, multiple myeloma / plasmatic cell neoplasia, myelodysplastic syndrome, myelogenous leukemia, myeloid leukemia, myeloproliferative disorders, paranasal sinus and nasal cavity cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma during pregnancy, nonmelanoma skin cancer, non-small cell lung cancer, metastatic squamous neck cancer with occult primary, buccopharyngeal cancer, malignant fibrous histiocytoma, malignant fibrous osteosarcoma / histiocytoma of the bone, epithelial ovarian cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, paraproteinemias, purpura, parathyroid cancer, cancer of the penis, phaeochromocytoma, hypophysis tumor, neoplasia of plasmatic cells / multiple myeloma, primary central nervous system lymphoma, primary liver cancer, prostate cancer, rectal cancer, renal cell cancer, cancer of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, cancer of the salivary glands, sarcoidosis, sarcomas, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous neck cancer, stomach cancer, pineal and supratentorial primitive neuroectodermal tumors, T-cell lymphoma, testicular cancer, thymoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, transitional renal pelvis and ureter cancer, trophoblastic tumors, cell cancer of the renal pelvis and ureter, cancer of the urethra, cancer of the uterus, uterine sarcoma, vaginal cancer, optic pathway and hypothalamic glioma, cancer of the vulva, Waldenstrom's macroglobulinemia, Wilms' tumor and any other hyperproliferative disease, as well as neoplasia, located in the system of a previously mentioned organ.

[0170] A “chemotherapy agent,” “chemotherapeutic,” “anti-cancer agent,” or the like, refer to compounds that are recognized to aid in the treatment of a cancer. Contemplated examples include the following agents or derivatives such as abemaciclib, abiraterone acetate, methotrexate, paclitaxel, adriamycin, acalabrutinib, brentuximab vedotin, ado-trastuzumab emtansine, aflibercept, afatinib, netupitant, palonosetron, imiquimod, aldesleukin, alectinib, alemtuzumab, pemetrexed disodium, copanlisib, melphalan, brigatinib, chlorambucil, amifostine, aminolevulinic acid, anastrozole, apalutamide, aprepitant, pamidronate disodium, exemestane, nelarabine, arsenic trioxide, ofatumumab, atezolizumab, bevacizumab, avelumab, axicabtagene ciloleucel, axitinib, azacitidine, carmustine, belinostat, bendamustine, inotuzumab ozogamicin, bevacizumab, bexarotene, bicalutamide, bleomycin, blinatumomab, bortezomib, bosutinib, brentuximab vedotin, brigatinib, busulfan, irinotecan, capecitabine, fluorouracil, carboplatin, carfilzomib, ceritinib, daunorubicin, cetuximab, cisplatin, cladribine, cyclophosphamide, clofarabine, cobimetinib, cabozantinib-S-malate, dactinomycin, crizotinib, ifosfamide, ramucirumab, cytarabine, dabrafenib, dacarbazine, decitabine, daratumumab, dasatinib, defibrotide, degarelix, denileukin diftitox, denosumab, dexamethasone, dexrazoxane, dinutuximab, docetaxel, doxorubicin, durvalumab, rasburicase, epirubicin, elotuzumab, oxaliplatin, eltrombopag olamine, enasidenib, enzalutamide, eribulin, vismodegib, erlotinib, etoposide, everolimus, raloxifene, toremifene, panobinostat, fulvestrant, letrozole, filgrastim, fludarabine, flutamide, pralatrexate, obinutuzumab, gefitinib, gemcitabine, gemtuzumab ozogamicin, glucarpidase, goserelin, propranolol, trastuzumab, topotecan, palbociclib, ibritumomab tiuxetan, ibrutinib, ponatinib, idarubicin, idelalisib, imatinib, talimogene laherparepvec, ipilimumab, romidepsin, ixabepilone, ixazomib, ruxolitinib, cabazitaxel, palifermin, pembrolizumab, ribociclib, tisagenlecleucel, lanreotide, lapatinib, olaratumab, lenalidomide, lenvatinib, leucovorin, leuprolide, lomustine, trifluridine, olaparib, vincristine, procarbazine, mechlorethamine, megestrol, trametinib, temozolomide, methylnaltrexone bromide, midostaurin, mitomycin C, mitoxantrone, plerixafor, vinorelbine, necitumumab, neratinib, sorafenib, nilutamide, nilotinib, niraparib, nivolumab, tamoxifen, romiplostim, sonidegib, omacetaxine, pegaspargase, ondansetron, osimertinib, panitumumab, pazopanib, interferon alfa-2b, pertuzumab, pomalidomide, mercaptopurine, regorafenib, rituximab, rolapitant, rucaparib, siltuximab, sunitinib, thioguanine, temsirolimus, thalidomide, thiotepa, trabectedin, valrubicin, vandetanib, vinblastine, vemurafenib, vorinostat, zoledronic acid, or combinations thereof such as cyclophosphamide, methotrexate, 5 -fluorouracil (CMF); doxorubicin, cyclophosphamide (AC); mustine, vincristine, procarbazine, prednisolone (MOPP); adriamycin, bleomycin, vinblastine, dacarbazine (ABVD); cyclophosphamide, doxorubicin, vincristine, prednisolone (CHOP); bleomycin, etoposide, cisplatin (BEP); epirubicin, cisplatin, 5- fluorouracil (ECF); epirubicin, cisplatin, capecitabine (ECX); methotrexate, vincristine, doxorubicin, cisplatin (MVAC). In certain embodiments, methods and compositions disclosed herein are used to treat or prevent and immune inflammatory related conditions such as rheumatoid arthritis, migraines, lupus, myositis, Sjogren’s syndrome, psoriasis, psoriatic arthritis, dermatomyositis, scleroderma, vasculitis, rheumatoid vasculitis, urticarial vasculitis, vitiligo, Crohn’s disease, celiac disease, ulcerative colitis, autoimmune gastritis, type 1 diabetes, Addison’s disease, Hashimoto’s thyroiditis, Graves’ disease, multiple sclerosis (MS), myasthenia gravis (MG), Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy (CIPD).

[0171] In certain embodiments, methods and compositions disclosed herein are used to treat or prevent cystic fibrosis comprising administering an effective amount of a vector(s) disclosed herein encoding a PD-L1 construct disclosed herein and a functional Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) to a subject in need thereof. In certain embodiments, the subject is diagnosed with a CFTR mutation. In certain embodiments administration is to the lung. In certain embodiments administration is by aerosol administration to the lung.

[0172] In certain embodiments, methods and compositions disclosed herein are used to treat or prevent hemophilia B comprising administering an effective amount of a vector(s) disclosed herein encoding a PD-L1 construct disclosed herein and Factor IX to a subject in need thereof. In certain embodiments administration is intramuscularly or into the hepatic artery.

[0173] In certain embodiments, methods and compositions disclosed herein are used to treat or prevent arthritis comprising administering an effective amount of a vector(s) disclosed herein encoding a PD-L1 construct disclosed herein and tumor necrosis factor receptor-immuno-globulin Fc (TNFR:Fc) fusion peptide to a subject in need thereof.

[0174] In certain embodiments, methods and compositions disclosed herein are used to treat or prevent hereditary emphysema comprising administering an effective amount of a vector(s) disclosed herein encoding a PD-L1 construct disclosed herein and alpha-1 antitrypsin to a subject in need thereof.

[0175] In certain embodiments, methods and compositions disclosed herein are used to treat or prevent Leber's congenital amaurosis comprising administering an effective amount of a vector(s) disclosed herein encoding a PD-L1 construct disclosed herein and a vector encoding the RPE65 protein (voretigene neparvovec) to a subject in need thereof. In certain embodiments, methods and compositions disclosed herein are used to treat or prevent muscular dystrophy comprising administering an effective amount of a vector(s) disclosed herein encoding a PD-L1 construct disclosed herein and alpha-sarcoglycan to a subject in need thereof.

[0176] In certain embodiments, methods and compositions disclosed herein are used to treat or prevent Parkinson's disease comprising administering an effective amount of a vector(s) disclosed herein encoding a PD-L1 construct disclosed herein and glutamate decarboxylase (65- and / or 67- kDa isoforms) of to a subject in need thereof.

[0177] In certain embodiments, methods and compositions disclosed herein are used to treat or prevent Alzheimer's disease comprising administering an effective amount of a vector(s) disclosed herein encoding a PD-L1 construct disclosed herein and nerve growth factor (NGF) to a subject in need thereof.

[0178] In certain embodiments, methods and compositions disclosed herein are used to treat or prevent spinal muscular atrophy comprising administering an effective amount of a vector(s) disclosed herein encoding a PD-L1 construct disclosed herein and survivor motor neuron 1 (onasemnogene abeparvovec) to a subject in need thereof.

[0179] In certain embodiments, methods and compositions disclosed herein are used to treat or prevent congestive heart failure comprising administering an effective amount of a vector(s) disclosed herein encoding a PD-L1 construct disclosed herein and sarco / endoplasmic reticulum Ca2+ ATPase (SERCA2a) to a subject in need thereof.

[0180] In certain embodiments, these agents may be administered in combination with antiinflammatory agents. Contemplated anti-inflammatory agents include both steroidal and nonsteroidal structures. Suitable non-limiting examples of steroidal anti-inflammatory compounds are corticosteroids such as hydrocortisone, cortisol, triamcinolone, alpha-methyl dexamethasone, dexamethasone-phosphate, beclomethasone dipropionates, clobetasol valerate, desonide, desoximetasone, desoxy corti costerone acetate, dexamethasone, diflorasone diacetate, diflucortolone valerate, fluadr enol one, fluclorolone acetonide, fludrocortisone, flumethasone pivalate, fluocinolone acetonide, fluocinonide, fluocortin butyl ester, fluocortolone, fluprednidene acetate, flurandrenolone, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone, fludrocortisone, fludrocortisone, fluocinolone, medrysone, betamethasone, chloroprednisone, dichlorisone, flunisolide, fluperolone, fluprednisolone, hydrocortisone valerate, hydrocortamate, prednisone, paramethasone, prednisolone, prednisone, beclomethasone dipropionate, and triamcinolone.

[0181] Pharmaceutical compositions

[0182] In certain embodiments, this disclosure relates to pharmaceutical compositions comprising vectors and other constructs disclosed herein. In certain embodiments, this disclosure relates to the production of a medicament comprising vectors and other constructs disclosed herein for use in treating or preventing a condition disclosed herein.

[0183] In certain embodiment, this disclosure contemplates pharmaceutical compositions comprising vectors and other constructs disclosed herein and pharmaceutically acceptable excipient. In certain embodiments, this disclosure contemplates the production of a medicament comprising vectors and other constructs disclosed herein and uses for methods disclosed herein.

[0184] Pharmaceutical compositions typically comprise an effective amount of vectors and other constructs disclosed herein and a suitable pharmaceutical acceptable carrier. The preparations can be prepared in a manner known per se, which usually involves mixing the vectors and other constructs according to the disclosure with the one or more pharmaceutically acceptable carriers, and, if desired, in combination with other pharmaceutical active compounds, when necessary under aseptic conditions. Reference is made to U.S. Pat. No. 6,372,778, U.S. Pat. No. 6,369,086, U.S. Pat. No. 6,369,087 and U.S. Pat. No. 6,372,733 and the further references mentioned above, as well as to the standard handbooks, such as the latest edition of Remington's Pharmaceutical Sciences.

[0185] In certain embodiments, the disclosure relates to pharmaceutical compositions comprising vectors and other constructs disclosed herein and a pharmaceutically acceptable excipient. In certain embodiments, the composition is an aqueous buffer, e.g., a pH between 6 and 8. Compositions suitable for injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), suitable mixtures thereof, vegetable (such as olive oil, sesame oil) and injectable organic esters such as ethyl oleate. Prevention of the action of microorganisms may be controlled by addition of any of various antibacterial and antifungal agents, example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0186] Providing a pharmaceutic composition is possible in a one-step process, simply by adding a suitable pharmaceutically acceptable diluent to vectors and other constructs disclosed herein in a container. In certain embodiments, the container is preferably a syringe for administering the reconstituted pharmaceutical composition after contact with the diluent. In certain embodiments, the vector constructs can be fdled into a syringe, and the syringe can then be closed with the stopper. A diluent is used in an amount to achieve the desired end-concentration. The pharmaceutical composition may contain other useful component, such as ions, buffers, excipients, stabilizers, etc.

[0187] In certain embodiments, this disclosure contemplates kits comprising vector constructs disclosed herein and a pharmaceutical composition comprising vectors and other constructs disclosed herein and a container with a suitable diluent. Further components of the kit may be instructions for use, administration means, such as syringes, catheters, brushes, etc. (if the compositions are not already provided in the administration means) or other components necessary for use in medical (surgical) practice, such as substitute needles or catheters, extra vials or further wound cover or bandage. In certain embodiments, the kit comprises a syringe housing the dry and stable hemostatic composition and a syringe containing the diluent (or provided to take up the diluent from another diluent container).

[0188] Co-expressed AAV-vectored PD-L1 improves the expression and protective efficacy of AAV- vectored bNAbs by reducing host anti-bNAb immune responses in rhesus macaques

[0189] Sustained expression of adeno-associated virus (AAV)-vectored anti -HIV- 1 broadly neutralizing antibodies (bNAbs) are contemplated for preventing and treating HIV-1 infection. Durable expression of AAV-vectored bNAbs in vivo is limited by host immune responses. PD-1 is an immune checkpoint protein on T cells that inhibits T-cell activity upon binding to PD-L1. PD- L1 co-expression on AAV.bNAb-transduced cells maintains bNAb expression by suppressing host anti-drug antibodies (ADA) and T-cell responses. A model was developed for prolonged AAV- vectored bNAb production in NHPs in order to advance AAV-delivery of bNAbs for human clinical trials. Data indicates that co-expressed AAV-vectored PD-L1 improves the expression and protective efficacy of AAV-vectored bNAbs by reducing host anti-bNAb immune responses in rhesus macaques.

Claims

CLAIMS1. A composition comprising a vector encoding PD-L1 in operable combination with a promoter and a vector encoding a viral binding agent in operable combination with a promoter.

2. The composition of claim 1, wherein the PD-L1 has a PD-L1 domain (DI) comprising the amino acid sequence ofFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEE DLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKV NA (SEQ ID NO: 8).

3. The composition of claim 1, wherein the vector encoding PD-L1 comprises MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQF VHGEEDLKVQHS S YRQRARLLKDQL SLGNAALQITD VKLQD AGVYR CMIS YGGAD YKRIT VK VNAP YNKINQRILVVDP VTSEHELTCQ AEGYPKAEVIWT S SDH QVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHP PNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET (SEQ ID NO: 6) or variants thereof.

4. The composition of claim 1, wherein the vector encoding PD-L1 and the vector encoding the viral binding agent is a recombinant adeno-associated virus (AAV).

5. The composition of claim 1, wherein the encoded viral binding agent is an anti -HIV antibody or binding fragment.

6. The composition of claim 5, wherein the antibody or binding fragment contains an antibody light chain and antibody heavy chain encoded by a single polypeptide connected by a peptide that generates polyproteins.

7. The composition of claim 6, wherein the peptide that generates polyproteins has the amino acid sequence of DXEXNPGP (SEQ ID NO: 1), wherein X is individually and independently at each occurrence any amino acid.

8. The composition of claim 6, wherein the peptide that generates polyproteins is (P2A) GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 2).

9. The composition of claim 1, wherein the vector encoding the viral binding agent in operable combination with a promoter is a chicken P-actin (CBA) promoter.

10. The composition of claim 9, wherein a SV40 intron is downstream of the CBA promoter, and a woodchuck post regulatory element (WPRE) is downstream of the encoded antibody heavy chain and antibody light chain.

11. The composition of claim 1, wherein the vector encoding PD-L1 in operable combination with a promoter is a cytomegalovirus (CMV) promoter and wherein the vector encoding the vector encoding the viral binding agent in operable combination with a promoter is a chicken P-actin (CBA) promoter.

12. The composition of claim 11, wherein a SV40 intron is downstream of the CMV promoter and a WPRE is downstream of the encoded PD-L1.

13. A method of treating HIV comprising administering an effective amount of a vector encoding PD-L1 in operable combination with a promoter and a vector encoding an HIV binding agent in operable combination with a promoter an any of claims 1-12 to a subject in need thereof.

14. A composition comprising a single vector encoding PD-L1 and encoding a viral binding agent in operable combination with a bidirectional enhancer.

15. The composition of claim 14, wherein the single vector encoding PD-L1 and encoding an HIV viral binding agent is a recombinant adeno-associated virus (AAV).

16. The composition of claim 15, wherein the recombinant adeno-associated virus (AAV) is a recombinant adeno-associated virus 9 (AAV9).

17. The composition of claim 14, wherein PD-L1 comprises a signal peptide, variable immunoglobulin motif, constant immunoglobulin motif, a transmembrane region and a cytoplasmic tail, and wherein a segment of or all of the constant immunoglobulin motif is deleted.

18. The composition of claims 14, wherein a segment or all of the cytoplasmic tail is deleted.

19. The composition of claim 18, wherein the segment of the cytoplasmic tail deleted is the C- terminal DTSSK (SEQ ID NO: 3) peptide sequence.

20. A method of treating HIV comprising administering an effective amount of a compositions comprising a vector encoding PD-L1 and encoding an HIV binding agent as in any of 14-19 to a subject in need thereof.

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