Anti-PD-1 antibody and uses thereof

Abstract

Provided herein are, inter alia, engineered antibodies (e.g., humanized antibodies, monoclonal antibodies) and antibody compositions (e.g., chimeric antigen receptors, bispecific antibodies) that are capable of binding PD-1. The engineered antibodies and antibody compositions provided herein include modified glycosylation patterns and lack an endoplasmic reticulum retention signal. The engineered antibodies bind PD-1 with high efficiency and specificity, thereby effectively targeting PD-1 expressing cells.

Description

Docket No.062822-501001WO PATENT APPLICATION ANTI-PD-1 ANTIBODY AND USES THEREOF Inventor(s): Waranyoo PHOOLCHAROEN Thailand Thailand Suthira TAYCHAKHOONAVUDH Thailand Thailand Assignee: Baiya Phytopharm Co., Ltd. Thailand Entity Size: SmallDocket No.062822-501001WO ANTI-PD-1 ANTIBODY AND USES THEREOF CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional App. Ser. No.63 / 583,056 filed on September 15, 2023, which is incorporated by reference herein in its entirety. REFERENCE TO A SEQUENCE LISTING

[0002] The Sequence Listing written in file “2024-09-11 Sequence_Listing_ST26062822- 501001WO.xml”, created on September 11, 2024, in XML format is 36 kb, and is hereby incorporated by reference in its entirety for all purposes. BACKGROUND

[0003] Monoclonal antibodies (mAbs) are one of the most widely used drug platforms for infectious diseases or cancer therapeutics because they selectively target pathogens, infectious cells, cancerous cells, and even immune cells.

[0004] Protein glycosylation is an essential co- and post-translational modification of secretory and membrane proteins in all eukaryotes. N-glycosylation profoundly affects the biological stability and function of therapeutic proteins, which explains the interest in glycoengineering technologies as methods to develop biobetter therapeutics. In current manufacturing processes, N-glycosylation is host-specific and remains difficult to control in a production environment that changes with scale and production batches leading to glycosylation heterogeneity and inconsistency. All non-engineered eukaryotic cells share a common N-glycosylation core, while terminal and lateral oligosaccharide residues are specific to each expression system. The processing of protein N-glycosylation within the cell endomembrane system is complex and often results in the production of a heterogeneous population of glycoforms.

[0005] The initial steps of N-glycosylation and N-glycan processing are highly conserved between plants, mammals, and yeast. In recent years, plants have gained increasing attention as manufacturing hosts for recombinant proteins, particularly for glycoprotein production including different human immunoglobulin subtypes or enzymes for treatment of lysosomal storage diseases. While mammalian expression systems like Chinese hamster ovary (CHO) cells are still widely used in the industry, plants provide an interesting alternative to the established hosts. The processing of protein N-glycosylation within the cell endomembrane system is complex and often results in the production of a heterogeneous population of glycoforms. The presence of signal peptide and KDEL sequence redirects the peptide to undergo subcellular localization in the secretory pathway with various glycosylation intermediary structures.Docket No.062822-501001WO

[0006] Several manufacturing approaches have been considered to modify the host N-glycosylation machinery, with the goal of exploiting the benefit of specific glycosylation profiles and generating therapeutics with targeted biological functions. These include modulation of cell growth conditions, silencing or overexpression of glycan processing enzymes, complete or partial removal of glycans, and in vitro glycosylation remodeling. While glycan engineering technologies using a α-1,6-fucosyltransferase (FUT8) knockout Chinese hamster ovary (CHO) cell line or plant α-1,3-fucosyltransferase / β-1,2- xylosyltransferase (∆FT / XT) knockdown / glycan engineered lines significantly reduced glycosylation heterogeneity, there remained a significant degree of non-uniformity in the final product. Protein glycosylation is considered a crucially important quality attribute, because of its major role in immunogenicity and clinical efficacy of therapeutic proteins. Additionally, these N-glycans are crucial for the structural integrity of the FcγR-binding site, and their removal has been shown to abrogate FcγR affinity and complement binding / effector functions especially reducing the ADCC or no ADCC. They also have an impact on the faster clearance rates of the drug with enhanced distribution rates.

[0007] One major advantage of plants as host cells is their comparable simple N-glycan processing pathway and the complete absence of mammalian-type O-glycosylation. However, the intrinsic biological variability of glycan structures creates a significant challenge for the plant platform. BRIEF SUMMARY

[0008] In one aspect, a recombinant antibody specific to PD-1 is provided, comprising one or more GlcNAc2Man3GlcNAc2 (GnGn) glycosylations.

[0009] In one aspect, a recombinant antibody specific to PD-1 is provided, comprising one or more antibody polypeptide sequences and a glycosylation pattern according to FIG.1B.

[0010] In one aspect, a recombinant antibody specific to PD-1 produced by expressing one or more antibody polypeptide sequences in a plant is provided, wherein the antibody comprises one or more GlcNAc2Man3GlcNAc2 (GnGn) glycosylations.

[0011] In some embodiments, the glycosylation is a N-glycosylation. In some embodiments, the glycosylation is at each Asn 297 in the Fc region of the antibody. In some embodiments, the glycosylation is essentially free of fucose. In some embodiments, the antibody comprises an Fc region selected from the group consisting of an IgGl, IgG2, IgG3 and IgG4 region.

[0012] In some embodiments, the antibody polypeptide sequences lack an endoplasmic reticulum retention signal. In some embodiments, the antibody polypeptide sequences comprises an endoplasmic reticulum retention signal and the endoplasmic reticulum retention signal is SEKDEL.

[0013] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is pembrolizumab. In some embodiments, the pembrolizumab comprises complementarityDocket No.062822-501001WO determining regions, heavy chain variable domain (VH), light chain variable domain (VL), or combinations thereof as described in Table 1.

[0014] In some embodiments, the antibody has an increased serum half-life in a mammal compared to any antibody produced by expressing polypeptides having an endoplasmic reticulum retention signal or produced in a mammalian expression system.

[0015] In one aspect, a method of making an antibody in plants is provided, comprising expressing in a plant one or more antibody polypeptide sequences that lack an endoplasmic reticulum retention signal.

[0016] In some embodiments, the plant is Nicotiana benthamiana. In some embodiments, the plant is not genetically engineered to alter glycosylation. In some embodiments, the plant is a wild-type plant. In some embodiments, the plant is a wild-type Nicotiana benthamiana. In some embodiments, the plant is genetically engineered to alter glycosylation. In some embodiments, the plant is a glycoengineered Nicotiana benthamiana. In some embodiments, the plant is ΔXF Nicotiana benthamiana.

[0017] In one aspect, a method of treating or preventing a condition associated with PD-1 function and / or of inhibiting PD-1 function in vivo is provided, comprising administering a subject an antibody of the disclosure.

[0018] In some embodiments, the antibody exhibits increased half-life in the subject compared to an antibody produced by expressing polypeptides having an endoplasmic reticulum retention signal or produced in a mammalian expression system. In some embodiments, the serum concentration of antibody in the subject is increased at 4 hours, 24 hours, or 48 hours compared to an antibody produced by expressing polypeptides having an endoplasmic reticulum retention signal, or produced in a mammalian expression system. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0020] FIG.1A shows a glycovariant antibody structure and expected glycosylation profile of plant- produced S-BPM-001-ΔXF. The pembrolizumab antibody (BPM-001) contains a signal peptide (S) at the N-terminus of HC and LC and co-expressed in glycoengineered N. benthamiana (ΔXF).

[0021] FIG.1B shows N-glycosylation in ΔXF plants produce humanized GnGn glycans on proteins. Black box, N-acetylglucosamine; white circle, mannose.

[0022] FIG.2 the left panel shows a glycovariant antibody structure and expected glycosylation profile of plant-produced S-BPM-001-WT. The pembrolizumab antibody (BPM-001) contains a signalDocket No.062822-501001WO peptide (S) at the N-terminus of HC and LC and co-expressed in wild-type N. benthamiana (WT). The right panel shows N-glycosylation in WT plants produce plant-specific β1,2-xylose (white star) and core α1,3-fucose (black triangle) glycans on proteins. Black box, N-acetylglucosamine; white circle, mannose.

[0023] FIG.3 shows a glycovariant antibody structure and expected glycosylation profile of plant- produced S-BPM-001-KD-WT. The pembrolizumab antibody (BPM-001) contains a signal peptide (S) at the N-terminus of HC and LC and SEKDEL at the C-terminus of HC. The HC and LC genes were co- expressed in wild-type N. benthamiana (WT). N-glycosylation of proteins with ER-retention signal SEKDEL in WT plants display mostly high mannose (white circle) glycans. Black box, N- acetylglucosamine.

[0024] FIG.4. shows a non-glycovariant antibody structure of plant-produced S-BPM-001-N297A- WT (left) and S-BPM-001-N297A-KD-WT (right). The pembrolizumab antibody (BPM-001) contains a signal peptide (S) at the N-terminus of HC and LC and an N297A mutation at the HC. The HC and LC genes were co-expressed in wild-type N. benthamiana (WT). N-glycosylation of protein with a mutated glycan site in WT plants lacks glycan structures.

[0025] FIG.5. shows a restriction enzyme digestion of variable and constant regions of BPM-001 (left) and pBaiYa vector (right). M, marker; Lane 1, S-BPM-001-VL; Lane 2, IgG4-CL; Lane 3, S-BPM- 001-VH; Lane 4, IgG4-CH; Lane 5, pBaiYa.

[0026] FIG.6 shows a PCR amplification of E. coli colonies containing BPM-001 LC and HC. M, marker; Lane 1, S-BPM-001-LC; Lane 2, S-BPM-001-HC; Lane 3, S-BPM-001-HC-KD.

[0027] FIG.7 shows a restriction enzyme digestion of BPM-001 LC and HC (left) and PCR amplification of A. tumefaciens clones containing HC and LC plasmids (right). M, marker; Lane 1, BPM-001 LC; Lane 2, BPM-001 HC; Lane 3, BPM-001-HC-KD.

[0028] FIG.8 shows an SDS-PAGE and Western blot of different plant-produced BPM-001 glycovariants. M, marker; Lane 1, Keytruda®; Lane 2, S-BPM-001-WT; Lane 3, S-BPM-001-KD-WT; Lane 4, S-BPM-001-ΔXF. Black arrow indicates the major bands for full antibody, HC and LC.

[0029] FIG.9 shows an SDS-PAGE of plant-produced S-BPM-001-N297-WT and S-BPM-001-N297- KD glycovariants. M, marker; Lane C, crude extract; Lane F, flow-through; Lane W, wash fraction; Lane E, elute fraction. Black arrow indicates the major bands for full antibody.

[0030] FIG.10 shows SDS-PAGE data for normalization of concentration (top) and binding profile of plant-produced BPM-001 glycovariants (bottom). All antibodies were diluted to 0.2 mg / mL and re- measured using the Bradford assay. The calculated concentrations were used for both SDS-PAGE and ELISA. Human PD-1 was coated and incubated with serial dilutions of BPM-001 antibodies and Keytruda®. An anti-Kappa chain antibody was used for detection.Docket No.062822-501001WO

[0031] FIG.11 shows N-glycan profiles of BPM-001 Pembrolizumab with different glycans analyzed by LC-MS. Chromatogram of EEQFNSTYR peptide showing peaks of different plant N-glycan attachments.

[0032] FIG.12 shows a pharmacokinetics (PK) study design in mice and repeated blood sampling approach for 7 days.

[0033] FIG.13 shows concentration-time curves of BPM-001 with different glycans after a single dose administration at 5 mg / kg in mice. Inset table comparing glycan patterns among the different glycovariants.

[0034] FIG.14 shows a graph of the half-life a of BPM-001 with different glycans calculated at 48 hrs.

[0035] FIGs.15A and B show half-life comparison of Keytruda vs S-BPM-001-ΔXF using Indirect (FIG.15A) or Sandwich ELISA (FIG.15B).

[0036] FIGs.16A-16E shows the translation of DNA to protein sequence for S-BPM-001-LC (FIG. 16A), S-BPM-001-HC (FIG.16B), and S-BPM-001-HC-KD (FIG.16C), and S-BPM-001-N297A HC- KD (FIG.16D) and S-BPM-001-N297A HC (FIG.16E). Protein sequences start at the first ATG of the DNA sequence. DETAILED DESCRIPTION

[0037] The disclosure is based in part on the discovery antibodies specific to PD-1 can be produced in plant cells with modified glycosylation patterns. The plant produced anti-PD-1 antibodies with modified glycosylation unexpectedly showed a longer half life in mammalian subjects after administration. The disclosure also provides methods for making the plant produced anti-PD-1 antibodies with modified glycosylation.

[0038] While various embodiments and aspects of the present invention are shown and described herein, it will be obvious to those skilled in the art that such embodiments and aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

[0039] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in the application including, without limitation, patents, patent applications, articles, books, manuals, and treatises are hereby expressly incorporated by reference in their entirety for any purpose.Docket No.062822-501001WO I. DEFINITIONS

[0040] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0041] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. See, e.g., Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this invention. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0042] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, and complements thereof. The term "polynucleotide" refers to a linear sequence of nucleotides. The term "nucleotide" typically refers to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof. Examples of polynucleotides contemplated herein include single and double stranded DNA, single and double stranded RNA (including siRNA), and hybrid molecules having mixtures of single and double stranded DNA and RNA. Nucleic acid as used herein also refers to nucleic acids that have the same basic chemical structure as a naturally occurring nucleic acid. Such analogues have modified sugars and / or modified ring substituents, but retain the same basic chemical structure as the naturally occurring nucleic acid. A nucleic acid mimetic refers to chemical compounds that have a structure that is different the general chemical structure of a nucleic acid, but that functions in a manner similar to a naturally occurring nucleic acid. Examples of such analogues include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).

[0043] The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group,Docket No.062822-501001WO e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0044] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0045] The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

[0046] An amino acid or nucleotide base "position" is denoted by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N- terminus (or 5'-end). Due to deletions, insertions, truncations, fusions, and the like that may be taken into account when determining an optimal alignment, in general the amino acid residue number in a test sequence determined by simply counting from the N-terminus will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, in a case where a variant has a deletion relative to an aligned reference sequence, there will be no amino acid in the variant that corresponds to a position in the reference sequence at the site of deletion. Where there is an insertion in an aligned reference sequence, that insertion will not correspond to a numbered amino acid position in the reference sequence. In the case of truncations or fusions there can be stretches of amino acids in either the reference or aligned sequence that do not correspond to any amino acid in the corresponding sequence.

[0047] The terms "numbered with reference to" or "corresponding to," when used in the context of the numbering of a given amino acid or polynucleotide sequence, refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. An amino acid residue in a protein "corresponds" to a given residue when it occupies the same essential structural position within the protein as the given residue. For example, a selected residue in a selected antibody (or Fab domain) corresponds to light chain threonine at Kabat position 40, when the selected residue occupies the same essential spatial or other structural relationship as a light chain threonine at Kabat position 40. In some embodiments, where a selected protein is aligned for maximum homology with the light chain of an antibody (or Fab domain), the position in the aligned selected protein aligning with threonine 40 is said to correspond to threonine 40. Instead of a primary sequence alignment, a three dimensional structural alignment can also be used, e.g., where the structure ofDocket No.062822-501001WO the selected protein is aligned for maximum correspondence with the light chain threonine at Kabat position 40, and the overall structures compared. In this case, an amino acid that occupies the same essential position as threonine 40 in the structural model is said to correspond to the threonine 40 residue.

[0048] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, "conservatively modified variants" refers to those nucleic acids that encode identical or essentially identical amino acid sequences. Because of the degeneracy of the genetic code, a number of nucleic acid sequences will encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0049] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.

[0050] The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).Docket No.062822-501001WO

[0051] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity over a specified region, e.g., of the entire polypeptide sequences of the invention or individual domains of the polypeptides of the invention), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be "substantially identical." This definition also refers to the complement of a test sequence. Optionally, the identity exists over a region that is at least about 50 nucleotides in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length.

[0052] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0053] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0054] A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of, e.g., a full length sequence or from 20 to 600, about 50 to about 200, or about 100 to about 150 amino acids or nucleotides in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math.2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol.48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat’l. Acad. Sci. USA 85:2444, by computerized implementations of theseDocket No.062822-501001WO algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

[0055] An example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res.25:3389-3402, and Altschul et al. (1990) J. Mol. Biol.215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0056] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0057] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodiesDocket No.062822-501001WO raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

[0058] Antibodies are large, complex molecules (molecular weight of ~150,000 or about 1320 amino acids) with intricate internal structure. A natural antibody molecule contains two identical pairs of polypeptide chains, each pair having one light chain and one heavy chain. Each light chain and heavy chain in turn consists of two regions: a variable (“V”) region, involved in binding the target antigen, and a constant (“C”) region that interacts with other components of the immune system. The light and heavy chain variable regions (also referred to herein as light chain variable (VL) domain and heavy chain variable (VH) domain, respectively) come together in 3-dimensional space to form a variable region that binds the antigen (for example, a receptor on the surface of a cell). Within each light or heavy chain variable region, there are three short segments (averaging 10 amino acids in length) called the complementarity determining regions (“CDRs”). The six CDRs in an antibody variable domain (three from the light chain and three from the heavy chain) fold up together in 3-dimensional space to form the actual antibody binding site which docks onto the target antigen. The position and length of the CDRs have been precisely defined by Kabat, E. et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1983, 1987. The part of a variable region not contained in the CDRs is called the framework ("FR"), which forms the environment for the CDRs.

[0059] An “antibody variant” as provided herein refers to a polypeptide capable of binding to an antigen and including one or more structural domains (e.g., light chain variable domain, heavy chain variable domain) of an antibody or fragment thereof. Non-limiting examples of antibody variants include single-domain antibodies or nanobodies, monospecific Fab2, bispecific Fab2, trispecific Fab3, monovalent IgGs, scFv, bispecific antibodies, bispecific diabodies, trispecific triabodies, scFv-Fc, minibodies, IgNAR, V-NAR, hcIgG, VhH, or peptibodies. A “peptibody” as provided herein refers to a peptide moiety attached (through a covalent or non-covalent linker) to the Fc domain of an antibody. Further non-limiting examples of antibody variants known in the art include antibodies produced by cartilaginous fish or camelids. A general description of antibodies from camelids and the variable regions thereof and methods for their production, isolation, and use may be found in references WO97 / 49805 and WO 97 / 49805 which are incorporated by reference herein in their entirety and for all purposes. Likewise, antibodies from cartilaginous fish and the variable regions thereof and methods for their production,Docket No.062822-501001WO isolation, and use may be found in WO2005 / 118629, which is incorporated by reference herein in its entirety and for all purposes.

[0060] The terms "CDR1", "CDR2" and "CDR3" as provided herein refer to the complementarity determining regions (CDR) 1, 2, and 3 of the light chain or heavy chain. "CDR L1", "CDR L2" and "CDR L3" as provided herein refer to the complementarity determining regions (CDR) 1, 2, and 3 of the variable light (L) chain of an antibody. In embodiments, the variable light chain provided herein includes in N-terminal to C-terminal direction a CDR L1, a CDR L2 and a CDR L3. Likewise, the terms "CDR H1", "CDR H2" and "CDR H3" as provided herein refer to the complementarity determining regions (CDR) 1, 2, and 3 of the variable heavy (H) chain of an antibody. In embodiments, the variable heavy chain provided herein includes in N-terminal to C-terminal direction a CDR H1, a CDR H2 and a CDR H3.

[0061] The terms "FR L1", "FR L2", "FR L3" and "FR L4" as provided herein are used according to their common meaning in the art and refer to the framework regions (FR) 1, 2, 3 and 4 of the variable light (L) chain of an antibody. In embodiments, the variable light chain provided herein includes in N- terminal to C-terminal direction a FR L1, a FR L2, a FR L3 and a FR L4. Likewise, the terms "FR H1", "FR H2", "FR H3" and "FR H4" as provided herein are used according to their common meaning in the art and refer to the framework regions (FR) 1, 2, 3 and 4 of the variable heavy (H) chain of an antibody. In embodiments, the variable heavy chain provided herein includes in N-terminal to C-terminal direction a FR H1, a FR H2, a FR H3 and a FR H4.

[0062] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL), variable light chain (VL) domain or light chain variable region and variable heavy chain (VH), variable heavy chain (VH) domain or heavy chain variable region refer to these light and heavy chain regions, respectively. The terms variable light chain (VL), variable light chain (VL) domain and light chain variable region as referred to herein may be used interchangeably. The terms variable heavy chain (VH), variable heavy chain (VH) domain and heavy chain variable region as referred to herein may be used interchangeably. The Fc (i.e. fragment crystallizable region) is the "base" or "tail" of an immunoglobulin and is typically composed of two heavy chains that contribute two or three constant domains depending on the class of the antibody. By binding to specific proteins, the Fc region ensures that each antibody generates an appropriate immune response for a given antigen. The Fc region also binds to various cell receptors, such as Fc receptors, and other immune molecules, such as complement proteins. The “Fc” fragment comprises the carboxy-terminal portions of both H chains held together byDocket No.062822-501001WO disulfides. The effector functions of antibodies are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain types of cells. Amino acid residue Asn 297 on the Fc domain can be modified by oligosaccharides attached to the Asn residue, termed glycosylation. This conserved glycosylation site on the Fc region of antibodies can be a target for modulation of antibody effector functions.

[0063] The term "antibody" is used according to its commonly known meaning in the art. Antibodies exist, e.g., as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH- CH1 by a disulfide bond. The F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)'2 dimer into an Fab' monomer. The Fab' monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed.1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990)). The term “antibody” as referred to herein further includes antibody variants such as single domain antibodies. Thus, in embodiments an antibody includes a single monomeric variable antibody domain. Thus, in embodiments, the antibody, includes a variable light chain (VL) domain or a variable heavy chain (VH) domain. In embodiments, the antibody is a variable light chain (VL) domain or a variable heavy chain (VH) domain. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively.

[0064] For preparation of monoclonal or polyclonal antibodies, any technique known in the art can be used (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); Cole et al., pp.77-96 in Monoclonal Antibodies and Cancer Therapy (1985)). "Monoclonal" antibodies (mAb) refer to antibodies derived from a single clone. Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies. Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al., NatureDocket No.062822-501001WO 348:552-554 (1990); Marks et al., Biotechnology 10:779-783 (1992)). Preparation of monoclonal antibodies from plants or plant cells is described herein and for example in Phakam et al., Front. Plant Sci., Sec. Plant Biotechnology (2021), 12.

[0065] The epitope of a mAb is the region of its antigen to which the mAb binds. Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) binding of the other to the antigen. That is, a 1x, 5x, 10x, 20x or 100x excess of one antibody inhibits binding of the other by at least 30% but preferably 50%, 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 50:1495, 1990). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

[0066] A single-chain variable fragment (scFv) is typically a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide of 10 to about 25 amino acids. The linker may usually be rich in glycine for flexibility, as well as serine or threonine for solubility. The linker can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa.

[0067] For preparation of suitable antibodies of the invention and for use according to the invention, e.g., recombinant, monoclonal, or polyclonal antibodies, many techniques known in the art can be used (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pp.77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985); Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988); and Goding, Monoclonal Antibodies: Principles and Practice (2d ed.1986)). The genes encoding the heavy and light chains of an antibody of interest can be cloned from a cell, e.g., the genes encoding a monoclonal antibody can be cloned from a hybridoma and used to produce a recombinant monoclonal antibody. Gene libraries encoding heavy and light chains of monoclonal antibodies can also be made from hybridoma or plasma cells. Random combinations of the heavy and light chain gene products generate a large pool of antibodies with different antigenic specificity (see, e.g., Kuby, Immunology (3rd ed.1997)). Techniques for the production of single chain antibodies or recombinant antibodies (U.S. Patent 4,946,778, U.S. Patent No.4,816,567) can be adapted to produce antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized or human antibodies (see, e.g., U.S. Patent Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, Marks et al., Bio / Technology 10:779-783 (1992); Lonberg et al., Nature 368:856- 859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); and Lonberg & Huszar, Intern. Rev. Immunol.Docket No.062822-501001WO 13:65-93 (1995)). Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10:779-783 (1992)). Antibodies can also be made bispecific, i.e., able to recognize two different antigens (see, e.g., WO 93 / 08829, Traunecker et al., EMBO J.10:3655-3659 (1991); and Suresh et al., Methods in Enzymology 121:210 (1986)). Antibodies can also be heteroconjugates, e.g., two covalently joined antibodies, or immunotoxins (see, e.g., U.S. Patent No.4,676,980 , WO 91 / 00360; WO 92 / 200373; and EP 03089).

[0068] Methods for humanizing or primatizing non-human antibodies are well known in the art (e.g., U.S. Patent Nos.4,816,567; 5,530,101; 5,859,205; 5,585,089; 5,693,761; 5,693,762; 5,777,085; 6,180,370; 6,210,671; and 6,329,511; WO 87 / 02671; EP Patent Application 0173494; Jones et al. (1986) Nature 321:522; and Verhoyen et al. (1988) Science 239:1534). Humanized antibodies are further described in, e.g., Winter and Milstein (1991) Nature 349:293. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co- workers (see, e.g., Morrison et al., PNAS USA, 81:6851-6855 (1984), Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Morrison and Oi, Adv. Immunol., 44:65-92 (1988), Verhoeyen et al., Science 239:1534-1536 (1988) and Presta, Curr. Op. Struct. Biol.2:593-596 (1992), Padlan, Molec. Immun., 28:489-498 (1991); Padlan, Molec. Immun., 31(3):169-217 (1994)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Patent No.4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. For example, polynucleotides comprising a first sequence coding for humanized immunoglobulin framework regions and a second sequence set coding for the desired immunoglobulin complementarity determining regions can be produced synthetically or by combining appropriate cDNA and genomic DNA segments. Human constant region DNA sequences can be isolated in accordance with well known procedures from a variety of human cells.

[0069] A "chimeric antibody" is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and / or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growthDocket No.062822-501001WO factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.

[0070] Techniques for conjugating therapeutic agents to antibodies are well known (see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp.243-56 (Alan R. Liss, Inc.1985); Hellstrom et al., “Antibodies For Drug Delivery”in Controlled Drug Delivery (2ndEd.), Robinson et al. (eds.), pp.623- 53 (Marcel Dekker, Inc.1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review" in Monoclonal Antibodies ‘84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62:119-58 (1982)). As used herein, the term “antibody-drug conjugate” or “ADC” refers to a therapeutic agent conjugated or otherwise covalently bound to to an antibody.

[0071] A "therapeutic agent" as referred to herein, is a composition useful in treating or preventing a disease such as cancer (e.g., leukemia). In embodiments, the therpaeutic agent is an anti-cancer agent. “Anti-cancer agent” is used in accordance with its plain ordinary meaning and refers to a composition (e.g., compound, drug, antagonist, inhibitor, modulator) having antineoplastic properties or the ability to inhibit the growth or proliferation of cells. In embodiments, an anti-cancer agent is a chemotherapeutic. In embodiments, an anti-cancer agent is an agent identified herein having utility in methods of treating cancer. In embodiments, an anti-cancer agent is an agent approved by the FDA or similar regulatory agency of a country other than the USA, for treating cancer.

[0072] The phrase "specifically (or selectively) binds" to an antibody or "specifically (or selectively) immunoreactive with," when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein, often in a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and more typically more than 10 to 100 times background. Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies can be selected to obtain only a subset of antibodies that are specifically immunoreactive with the selected antigen and not with other proteins. This selection may be achieved by subtracting out antibodies that cross-react with other molecules. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).Docket No.062822-501001WO

[0073] As used herein, the terms “glycosylation,” “glycan,” and “glycoform” are used interchangeably and refer to an N-linked or O-linked oligosaccharide. An N-linked oligosaccharide for example is or was attached by an N-acetylglucosamine residue linked to the amide nitrogen of an asparagine (Asn, N) residue in a protein or polypeptide. The predominant sugars found on glycoproteins are glucose, galactose, mannose, fucose, N-acetylgalactosamine (GalΝAc), N- acetylglucosamine (GlcΝAc), and sialic acid (e.g., N-acetyl-neuraminic acid (NANA)). The processing of the sugar groups occurs co- translationally in the lumen of the ER and continues in the Golgi apparatus for N-linked glycoproteins. N- glycans can have a common pentasaccharide core of Man3GlcNAc2 ("Man" refers to mannose; "Glc" refers to glucose; and "NAc" refers to N-acetyl; GlcΝAc refers to N-acetylglucosamine). Ν-glycans differ with respect to the number of branches (antennae) comprising peripheral sugars (e.g., GlcΝAc, galactose, fucose, and sialic acid) that are added to the Man3GlcNAc2 ("Man3") core structure which is also referred to as the "trimannose core", the "pentasaccharide core" or the "paucimannose core". N-glycans can be classified according to their branched constituents (e.g., high mannose, complex or hybrid). A "high mannose" type N- glycan has five or more mannose residues. A "complex" type N-glycan typically has at least one GlcΝAc attached to the 1,3 mannose arm and at least one GlcΝAc attached to the 1,6 mannose arm of a "trimannose" core. Complex N-glycans may also have galactose ("Gal") or N- acetylgalactosamine ("GalΝAc") residues that are optionally modified with sialic acid or derivatives (e.g., "NANA" or "NeuAc", where "Neu" refers to neuraminic acid and "Ac" refers to acetyl). Complex N- glycans may also have intrachain substitutions comprising "bisecting" GlcΝAc and core fucose ("Fuc"). Complex N-glycans may also have multiple antennae on the "trimannose core," often referred to as "multiple antennary glycans." A "hybrid" N-glycan has at least one GlcΝAc on the terminal of the 1,3 mannose arm of the trimannose core and zero or more mannoses on the 1,6 mannose arm of the trimannose core. The various N-glycans are also referred to as "glycoforms." A "ligand" refers to an agent, e.g., a polypeptide or other molecule, capable of binding to a receptor or antibody, antibody variant, antibody region or fragment thereof. The glycan analysis and distribution on any Ig molecule can be determined by several mass spectroscopy methods known to one skilled in the art, including but not limited to: HPLC, NMR, LCMS, and MALDI-TOF MS.

[0074] A "label" or a "detectable moiety" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into a peptide or antibody specifically reactive with a target peptide. Any appropriate method known in the art for conjugating an antibody to the label may be employed, e.g., using methods described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego.Docket No.062822-501001WO

[0075] "Contacting" is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species (e.g., antibodies and antigens) to become sufficiently proximal to react, interact, or physically touch. It should be appreciated; however, that the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents which can be produced in the reaction mixture.

[0076] The term "contacting" may include allowing two species to react, interact, or physically touch, wherein the two species may be, for example, a pharmaceutical composition as provided herein and a cell. In embodiments contacting includes, for example, allowing a pharmaceutical composition as described herein to interact with a cell.

[0077] A "cell" as used herein, refers to a cell carrying out metabolic or other function sufficient to preserve or replicate its genomic DNA. A cell can be identified by well-known methods in the art including, for example, presence of an intact membrane, staining by a particular dye, ability to produce progeny or, in the case of a gamete, ability to combine with a second gamete to produce a viable offspring. Cells may include prokaryotic and eukaryotic cells. Prokaryotic cells include but are not limited to bacteria. Eukaryotic cells include, but are not limited to, yeast cells and cells derived from plants and animals, for example mammalian, insect (e.g., spodoptera) and human cells.

[0078] “T cells” or “T lymphocytes” as used herein are a type of lymphocyte (a subtype of white blood cell) that plays a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells, by the presence of a T-cell receptor on the cell surface. T cells include, for example, natural killer T (NKT) cells, cytotoxic T lymphocytes (CTLs), regulatory T (Treg) cells, and T helper cells. Different types of T cells can be distinguished by use of T cell detection agents.

[0079] As defined herein, the term "inhibition", "inhibit", "inhibiting" and the like in reference to a protein-inhibitor (e.g., an A2A receptor antagonist or a PD-1 signaling pathway inhibitor) interaction means negatively affecting (e.g., decreasing) the activity or function of the protein (e.g., decreasing the activity of an A2A receptor or a PD-1 protein or PD-L1 protein) relative to the activity or function of the protein in the absence of the inhibitor (e.g., an A2A receptor antagonist or a PD-1 signaling pathway inhibitor). In some embodiments, inhibition refers to reduction of a disease or symptoms of disease (e.g., cancer). Thus, inhibition includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signal transduction or enzymatic activity or the amount of a protein (e.g., an A2A receptor or a PD-1 protein or PD-L1 protein). Similarly an "inhibitor" is a compound or protein that inhibits an A2A receptor or a PD-1 protein or PD-L1 protein, e.g.,, by binding, partially or totally blocking, decreasing, preventing, delaying,Docket No.062822-501001WO inactivating, desensitizing, or down-regulating activity (e.g., an A2A receptor activity or a PD-1 protein activity or PD-L1 protein activity).

[0080] A "PD-1 protein" or "PD-1" as referred to herein includes any of the recombinant or naturally- occurring forms of the Programmed cell death protein 1 (PD-1) also known as cluster of differentiation 279 (CD 279) or variants or homologs thereof that maintain PD-1 protein activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to PD-1 protein). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring PD-1 protein. In embodiments, the PD-1 protein is substantially identical to the protein identified by the UniProt reference number Q15116 or a variant or homolog having substantial identity thereto. In embodiments, the PD-1 protein is substantially identical to the protein identified by the UniProt reference number Q02242 or a variant or homolog having substantial identity thereto. In embodiments, the PD-1 inhibitor is Nivolumab, Pembrolizumab, or Cemiplimab. In embodiments, the PD-1 inhibitor is Nivolumab. In embodiments, the PD-1 inhibitor is Pembrolizumab. In embodiments, the PD-1 inhibitor is Cemiplimab.

[0081] The term “cemiplimab” as provided herein refers to a monoclonal antibody capable of binding the programmed death receptor-1 (PD-1). In the usual and customary sense “cemiplimab” refers to the antibody identified by Cas Registry number 1801342-60-8.

[0082] The term “pembrolizumab” as provided herein refers to a humanized antibody capable of binding the programmed death receptor-1 (PD-1). In the usual and customary sense “pembrolizumab” refers to the antibody identified by Cas Registry number 1374853-91-4.

[0083] The term “nivolumab” as provided herein refers to a monoclonal antibody capable of binding the programmed death receptor-1 (PD-1). In the usual and customary sense “nivolumab” refers to the antibody identified by Cas Registry number 946414-94-4.

[0084] The term "recombinant" when used with reference, e.g., to a cell, nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all. Transgenic cells and plants are those that express a heterologous gene or coding sequence, typically as a result of recombinant methods.

[0085] The term "heterologous" when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or moreDocket No.062822-501001WO sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0086] The term "exogenous" refers to a molecule or substance (e.g., a compound, nucleic acid or protein) that originates from outside a given cell or organism. For example, an "exogenous promoter" as referred to herein is a promoter that does not originate from the cell or organism it is expressed by. Conversely, the term "endogenous" or "endogenous promoter" refers to a molecule or substance that is native to, or originates within, a given cell or organism.

[0087] As defined herein, the term "inhibition", "inhibit", "inhibiting" and the like in reference to cell proliferation (e.g., cancer cell proliferation) means negatively affecting (e.g., decreasing proliferation) or killing the cell. In some embodiments, inhibition refers to reduction of a disease or symptoms of disease (e.g., cancer, cancer cell proliferation). Thus, inhibition includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signal transduction or enzymatic activity or the amount of a protein. Similarly an "inhibitor" is a compound or protein that inhibits a receptor or another protein, e.g.,, by binding, partially or totally blocking, decreasing, preventing, delaying, inactivating, desensitizing, or down-regulating activity (e.g., a receptor activity or a protein activity).

[0088] “Biological sample” or “sample” refer to materials obtained from or derived from a subject or patient. A biological sample includes sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histological purposes. Such samples include bodily fluids such as blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, and the like), sputum, tissue, cultured cells (e.g., primary cultures, explants, and transformed cells) stool, urine, synovial fluid, joint tissue, synovial tissue, synoviocytes, fibroblast-like synoviocytes, macrophage-like synoviocytes, immune cells, hematopoietic cells, fibroblasts, macrophages, T cells, etc. A biological sample is typically obtained from a eukaryotic organism, such as a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish.

[0089] A “control” or “standard control” refers to a sample, measurement, or value that serves as a reference, usually a known reference, for comparison to a test sample, measurement, or value. For example, a test sample can be taken from a patient suspected of having a given disease (e.g., cancer) and compared to a known normal (non-diseased) individual (e.g., a standard control subject). A standard control can also represent an average measurement or value gathered from a population of similar individuals (e.g., standard control subjects) that do not have a given disease (i.e. standard control population), e.g., healthy individuals with a similar medical background, same age, weight, etc. ADocket No.062822-501001WO standard control value can also be obtained from the same individual, e.g., from an earlier-obtained sample from the patient prior to disease onset. For example, a control can be devised to compare therapeutic benefit based on pharmacological data (e.g., half-life) or therapeutic measures (e.g., comparison of side effects). Controls are also valuable for determining the significance of data. For example, if values for a given parameter are widely variant in controls, variation in test samples will not be considered as significant. One of skill will recognize that standard controls can be designed for assessment of any number of parameters (e.g., RNA levels, protein levels, specific cell types, specific bodily fluids, specific tissues, synoviocytes, synovial fluid, synovial tissue, fibroblast-like synoviocytes, macrophagelike synoviocytes, etc).

[0090] One of skill in the art will understand which standard controls are most appropriate in a given situation and be able to analyze data based on comparisons to standard control values. Standard controls are also valuable for determining the significance (e.g., statistical significance) of data. For example, if values for a given parameter are widely variant in standard controls, variation in test samples will not be considered as significant.

[0091] “Patient” or “subject in need thereof” refers to a living organism suffering from or prone to a disease or condition that can be treated by administration of a composition or pharmaceutical composition as provided herein. Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and other non-mammalian animals. In some embodiments, a patient is human.

[0092] The terms “disease” or “condition” refer to a state of being or health status of a patient or subject capable of being treated with the compounds or methods provided herein. The disease may be a cancer. In some further instances, “cancer” refers to human cancers and carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, including solid and lymphoid cancers, kidney, breast, lung, bladder, colon, ovarian, prostate, pancreas, stomach, brain, head and neck, skin, uterine, testicular, glioma, esophagus, and liver cancer, including hepatocarcinoma, lymphoma, including B-acute lymphoblastic lymphoma, non-Hodgkin’s lymphomas (e.g., Burkitt’s, Small Cell, and Large Cell lymphomas), Hodgkin’s lymphoma, leukemia (including acute myeloid leukemia (AML), ALL, and CML), or multiple myeloma.

[0093] As used herein, the term “cancer” refers to all types of cancer, neoplasm or malignant tumors found in mammals (e.g., humans), including leukemia, carcinomas and sarcomas. Exemplary cancers that may be treated with a compound or method provided herein include breast cancer, colon cancer, kidney cancer, leukemia, lung cancer, melanoma, ovarian cancer, prostate cancer, pancreatic cancer, brain cancer, liver cancer, gastric cancer or a sarcoma.Docket No.062822-501001WO

[0094] The term “leukemia” refers broadly to progressive, malignant diseases of the blood-forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is generally clinically classified on the basis of (1) the duration and character of the disease-acute or chronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid (lymphogenous), or monocytic; and (3) the increase or non-increase in the number abnormal cells in the blood-leukemic or aleukemic (subleukemic). Exemplary leukemias that may be treated with a compound or method provided herein include, for example, acute myeloid leukemia, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, multiple myeloma, plasmacytic leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, or undifferentiated cell leukemia.

[0095] The term “sarcoma” generally refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance. Sarcomas that may be treated with a compound or method provided herein include a chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, or telangiectaltic sarcoma.

[0096] The term “melanoma” is taken to mean a tumor arising from the melanocytic system of the skin and other organs. Melanomas that may be treated with a compound or method provided herein include, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman'sDocket No.062822-501001WO melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungal melanoma, or superficial spreading melanoma.

[0097] The term “carcinoma” refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases. Exemplary carcinomas that may be treated with a compound or method provided herein include, for example, medullary thyroid carcinoma, familial medullary thyroid carcinoma, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniforni carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypernephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, nasopharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, or carcinoma villosum.

[0098] As used herein, the terms "metastasis," "metastatic," and "metastatic cancer" can be used interchangeably and refer to the spread of a proliferative disease or disorder, e.g., cancer, from one organ or another non-adjacent organ or body part. Cancer occurs at an originating site, e.g., breast, which site is referred to as a primary tumor, e.g., primary breast cancer. Some cancer cells in the primary tumor or originating site acquire the ability to penetrate and infiltrate surrounding normal tissue in the local areaDocket No.062822-501001WO and / or the ability to penetrate the walls of the lymphatic system or vascular system circulating through the system to other sites and tissues in the body. A second clinically detectable tumor formed from cancer cells of a primary tumor is referred to as a metastatic or secondary tumor. When cancer cells metastasize, the metastatic tumor and its cells are presumed to be similar to those of the original tumor. Thus, if lung cancer metastasizes to the breast, the secondary tumor at the site of the breast consists of abnormal lung cells and not abnormal breast cells. The secondary tumor in the breast is referred to a metastatic lung cancer. Thus, the phrase metastatic cancer refers to a disease in which a subject has or had a primary tumor and has one or more secondary tumors. The phrases non-metastatic cancer or subjects with cancer that is not metastatic refers to diseases in which subjects have a primary tumor but not one or more secondary tumors. For example, metastatic lung cancer refers to a disease in a subject with or with a history of a primary lung tumor and with one or more secondary tumors at a second location or multiple locations, e.g., in the breast.

[0099] The term "associated" or "associated with" in the context of a substance or substance activity or function associated with a disease (e.g., cancer (e.g., leukemia, acute myeloid leukemia)) means that the disease (e.g., cancer (e.g., leukemia, acute myeloid leukemia)) is caused by (in whole or in part), or a symptom of the disease is caused by (in whole or in part) the substance or substance activity or function. For example, a disease can be asscoiated with PD-1 function and / or of inhibiting PD-1 function.

[0100] As used herein, “treating” or “treatment of” a condition, disease or disorder or symptoms associated with a condition, disease or disorder refers to an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of condition, disorder or disease, stabilization of the state of condition, disorder or disease, prevention of development of condition, disorder or disease, prevention of spread of condition, disorder or disease, delay or slowing of condition, disorder or disease progression, delay or slowing of condition, disorder or disease onset, amelioration or palliation of the condition, disorder or disease state, and remission, whether partial or total. “Treating” can also mean prolonging survival of a subject beyond that expected in the absence of treatment. “Treating” can also mean inhibiting the progression of the condition, disorder or disease, slowing the progression of the condition, disorder or disease temporarily, although in some instances, it involves halting the progression of the condition, disorder or disease permanently. As used herein the terms treatment, treat, or treating refers to a method of reducing the effects of one or more symptoms of a disease or condition characterized by expression of the protease or symptom of the disease or condition characterized by expression of the protease. Thus in the disclosed method, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease, condition, or symptom of the disease or condition. For example, a method for treating a disease isDocket No.062822-501001WO considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject as compared to a control. Thus the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition. Further, as used herein, references to decreasing, reducing, or inhibiting include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater as compared to a control level and such terms can include but do not necessarily include complete elimination.

[0101] The terms “dose” and “dosage” are used interchangeably herein. A dose refers to the amount of active ingredient given to an individual at each administration. The dose will vary depending on a number of factors, including the range of normal doses for a given therapy, frequency of administration; size and tolerance of the individual; severity of the condition; risk of side effects; and the route of administration. One of skill will recognize that the dose can be modified depending on the above factors or based on therapeutic progress. The term “dosage form” refers to the particular format of the pharmaceutical or pharmaceutical composition, and depends on the route of administration. For example, a dosage form can be in a liquid form for nebulization, e.g., for inhalants, in a tablet or liquid, e.g., for oral delivery, or a saline solution, e.g., for injection.

[0102] By “therapeutically effective dose or amount” as used herein is meant a dose that produces effects for which it is administered (e.g., treating or preventing a disease). The exact dose and formulation will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols.1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Remington: The Science and Practice of Pharmacy, 20th Edition, Gennaro, Editor (2003), and Pickar, Dosage Calculations (1999)). For example, for the given parameter, a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a standard control. A therapeutically effective dose or amount may ameliorate one or more symptoms of a disease. A therapeutically effective dose or amount may prevent or delay the onset of a disease or one or more symptoms of a disease when the effect for which it is being administered is to treat a person who is at risk of developing the disease.

[0103] As used herein, the term "administering" means oral administration, administration as a suppository, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-Docket No.062822-501001WO osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. By "co-administer" it is meant that a composition described herein is administered at the same time, just prior to, or just after the administration of one or more additional therapies, for example cancer therapies such as chemotherapy, hormonal therapy, radiotherapy, or immunotherapy. The compounds of the invention can be administered alone or can be coadministered to the patient. Coadministration is meant to include simultaneous or sequential administration of the compounds individually or in combination (more than one compound). Thus, the preparations can also be combined, when desired, with other active substances (e.g., to reduce metabolic degradation). The compositions of the present invention can be delivered by transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.

[0104] The compositions of the present invention may additionally include components to provide sustained release and / or comfort. Such components include high molecular weight, anionic mucomimetic polymers, gelling polysaccharides and finely-divided drug carrier substrates. These components are discussed in greater detail in U.S. Pat. Nos.4,911,920; 5,403,841; 5,212,162; and 4,861,760. The entire contents of these patents are incorporated herein by reference in their entirety for all purposes. The compositions of the present invention can also be delivered as microspheres for slow release in the body. For example, microspheres can be administered via intradermal injection of drug-containing microspheres, which slowly release subcutaneously (see Rao, J. Biomater Sci. Polym. Ed.7:623-645, 1995; as biodegradable and injectable gel formulations (see, e.g., Gao Pharm. Res.12:857-863, 1995); or, as microspheres for oral administration (see, e.g., Eyles, J. Pharm. Pharmacol.49:669-674, 1997). In embodiments, the formulations of the compositions of the present invention can be delivered by the use of liposomes which fuse with the cellular membrane or are endocytosed, i.e., by employing receptor ligands attached to the liposome, that bind to surface membrane protein receptors of the cell resulting in endocytosis. By using liposomes, particularly where the liposome surface carries receptor ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the compositions of the present invention into the target cells in vivo. (See, e.g., Al-Muhammed, J. Microencapsul.13:293-306, 1996; Chonn, Curr. Opin. Biotechnol.6:698-708, 1995; Ostro, Am. J. Hosp. Pharm.46:1576-1587, 1989). The compositions of the present invention can also be delivered as nanoparticles.Docket No.062822-501001WO

[0105] As used herein, the term “pharmaceutically acceptable” is used synonymously with “physiologically acceptable” and “pharmacologically acceptable”. A pharmaceutical composition will generally comprise agents for buffering and preservation in storage, and can include buffers and carriers for appropriate delivery, depending on the route of administration.

[0106] "Pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" refer to a substance that aids the administration of an active agent to and absorption by a subject and can be included in the compositions of the present invention without causing a significant adverse toxicological effect on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and / or aromatic substances and the like that do not deleteriously react with the compounds of the invention. One of skill in the art will recognize that other pharmaceutical excipients are useful in the present invention.

[0107] The term "pharmaceutically acceptable salt" refers to salts derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like.

[0108] The term "preparation" is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.

[0109] The pharmaceutical preparation is optionally in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form. The unit dosage form can be of a frozen dispersion.

[0110] A “synergistic amount” as used herein refers to the sum of a first amount (e.g., an amount of a compound provided herein) and a second amount (e.g., a therapeutic agent) that results in a synergisticDocket No.062822-501001WO effect (i.e. an effect greater than an additive effect). Therefore, the terms "synergy", "synergism", "synergistic", "combined synergistic amount", and "synergistic therapeutic effect" which are used herein interchangeably, refer to a measured effect of the compound administered in combination where the measured effect is greater than the sum of the individual effects of each of the compounds provided herein administered alone as a single agent.

[0111] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. II ANTIBODY COMPOSITIONS

[0112] Provided herein are, inter alia, antibodies and antibody compositions that are capable of binding PD-1. The antibodies include anti-PD-1 antibodies that have glycoengineered glycosylation patterns. The anti-PD-1 antibodies with glycoengineered glycosylation patterns inlcude antibodies that have altered half lifes in mammals compared to other previosuly known anti-PD-1 antibodies with different glycosylation patterns.

[0113] In one aspect, the disclosure provides for a recombinant antibody specific to PD-1, comprising one or more glycosylations.

[0114] In one aspect, the disclosure provides for a recombinant antibody specific to PD-1, comprising one or more GlcNAc2Man3GlcNAc2 (GnGn) glycosylations.

[0115] In one aspect, the disclosure provides for a recombinant antibody specific to PD-1, comprising one or more antibody polypeptide sequences and a glycosylation pattern according to FIG.1B.

[0116] In one aspect, the disclosure provides for a recombinant antibody specific to PD-1, produced by expressing one or more antibody polypeptide sequences in a plant, wherein the antibody comprises one or more glycosylations.

[0117] In one aspect, the disclosure provides for a recombinant antibody specific to PD-1, produced by expressing one or more antibody polypeptide sequences in a plant, wherein the antibody comprises one or more GlcNAc2Man3GlcNAc2 (GnGn) glycosylations.

[0118] Antibodies produced in mammalian cells can show many N-linked glycoforms for example as detected by mass spectrometric analyses on total protein extractions. Such heterogeneity makes a controlled production difficult and hinders batch to batch consistency, an important requirement of the regulatory authorities. Moreover, glycosylation heterogeneity impedes studies to determine the impact of individual glycoforms to a protein's function. Plant cells synthesize only a few N-glycan species. In plantDocket No.062822-501001WO cells, two glycoforms account for >90% of the glycosylated species. The major one (>60%) are GnGnXF structures, a complex N-glycan formation carrying β1,2-linked xylose and α1,3-linked fucose residues attached to a GlcNAc2Man3GlcNAc2 (GnGn) core. The second major glycoform (20–40%) are paucimannosidic structures, N-glycans that lack the two terminal GlcNAc residues at their non-reducing ends.

[0119] In some embodiments, the glycosylation is a N-Glycosylation. In some embodiments, the glycosylation is an O-Glycosylation. In some embodiments, the N-Glycosylation site has the consensus sequence NX(S / T), with X being any amino acid, except proline. In some embodiments, the glycosylation site is at an amino acid residue corresponding to N297 (EU numbering (Edelman et al., 1969) and (Kabat et al., Sequences of Proteins of Immunological Interest, 1991) ) in the CH2 region of IgG1. In some embodiments, the glycosylation is at each N297 (Asn297) in the Fc region of the antibody.

[0120] In some embodiments, the glycosylation comprises one or more galactose, mannose, fucose, N- acetylglucosamine, α(2,3) –linked N-acetylneuraminic acid, α(2,6) –linked N-acetylneuraminic acid, β1,2-xylose, or α1,3-fucose. In some embodiments, the glycosylation comprises one or more β1,2-xylose, α1,3-fucose, N-acetylglucosamine, or mannose.

[0121] In some embodiments, the glycosylation pattern is a complex N-glycosylation and comprises one β1,2-xylose, one α1,3-fucose, and / or one or two β-1,2-linked N-acetylglucosamine bound to a trimannosyl chitobiose core. In some embodiments, the glycosylation pattern is a high-mannose N- glycosylation and comprises two or more mannose bound to the core. In some embodiments, the glycosylation pattern is a humanized N-glycosylation and comprises one or two β-1,2-linked N- acetylglucosamine bound to the core and lacks β1,2-xylose and α1,3-fucose. In some embodiments, the glycosylation pattern comprises a GlcNAc2Man3GlcNAc2 (GnGn). In some embodiments, the glycosylation pattern is a GlcNAc2Man3GlcNAc2 (GnGn). In some embodiments, the glycosylation pattern is a GlcNAc2Man3GlcNAc2 (GnGn) and lacks β1,2-xylose and α1,3-fucose. In some embodiments, the glycosylation is essentially free of fucose.

[0122] In some embodiments, the endoplasmic reticulum retention signal is SEKDEL. In some embodiments, the antibody polypeptide sequence lacks an endoplasmic reticulum retention signal.

[0123] In some embodiments, the antibody is a monoclonal antibody.

[0124] In some embodiments, the antibody comprises an Fc region selected from the group consisting of an IgGl, IgG2, IgG3 and IgG4 region.

[0125] In some embodiments, the antibody is Pembrolizumab, Nivolumab, or Cemiplimab.

[0126] In some embodiments, the antibody is Pembrolizumab. Pembrolizumab is a humanized monoclonal IgG4 (S228P) antibody directed against human cell surface receptor PD-1 (programmedDocket No.062822-501001WO death- 1 or programmed cell death- 1). Pembrolizumab is described, for example, in U.S. Patent Nos. 8,354,509 and 8,900,587.

[0127] In some embodiments, the pembrolizumab comprises complementarity determining regions, heavy chain variable domain (VH), light chain variable domain (VL), or combinations thereof as described in Table 1. Table 1. Exemplary Antibody Sequences SEQ ID Description Sequence NO: 9 Pembrolizumab HC QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGG INPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRD YRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKT YTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 10 Pembrolizumab LC EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYL ASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC 11 Signal peptide Accession No. MGWSCIILFLVATATGVHS P01750 12 Part of restriction enzyme sequenceDVQLLE6 SP-Keytruda- MGWSCIILFLVATATGVHSDVQLLEEIVLTQSPATLSLSPGERATLSCRASKGV LC(IgG4) with STSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEP Signal peptide EDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC 7 SP-Keytruda- MGWSCIILFLVATATGVHSDVQLLEQVQLVQSGVEVKKPGASVKVSCKASGY HC(IgG4) with TFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTT Signal peptide TAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSV FPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 8 SP-Keytruda- MGWSCIILFLVATATGVHSDVQLLEQVQLVQSGVEVKKPGASVKVSCKASGYTFT HC(IgG4) with NYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYME Signal peptide LKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRS and SEKDEL TSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS Sequence SLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWDocket No.062822-501001WO QEGNVFSCSVMHEALHNHYTQKSLSLSLGKSEKDEL 13 Pembrolizumab HC QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMG Variable region GINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYR FDMGFDYWGQGTTVTVSS 14 Pembrolizumab LC EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLA Variable region SYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK 15 Pembrolizumab HC NYYMY CDR1 16 Pembrolizumab HC GINPSNGGTNFNEKFKN CDR2 17 Pembrolizumab HC RDYRFDMGFDY CDR3 18 Pembrolizumab LC RASKGVSTSGYSYLH CDR1 19 Pembrolizumab LC LASYLES CDR2 20 Pembrolizumab LC QHSRDLPLT CDR3 21 SP-Keytruda- MGWSCIILFLVATATGVHSDVQLLEQVQLVQSGVEVKKPGASVKVSCKASGY HC(IgG4) with TFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTT Signal peptide TAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSV N297A FPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA KTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0128] In some embodiments, the antibody comprises light and heavy chain variable domains.

[0129] In some embodiments, the light chain variable domain includes: a CDR1 as set forth in SEQ ID NO:18, a CDR2 as set forth in SEQ ID NO:19, a CDR3 as set forth in SEQ ID NO:20. In some embodiments, the heavy chain variable domain includes: a CDR1 as set forth in SEQ ID NO:15, a CDR2 as set forth in SEQ ID NO:16, and a CDR3 as set forth in SEQ ID NO:17. In some embodiments, the light chain variable domain includes: a CDR1 as set forth in SEQ ID NO:18, a CDR2 as set forth in SEQ ID NO:19, a CDR3 as set forth in SEQ ID NO:20, and the heavy chain variable domain includes: a CDR1 as set forth in SEQ ID NO:15, a CDR2 as set forth in SEQ ID NO:16, and a CDR3 as set forth in SEQ ID NO:17.

[0130] In some embodiments, the light chain variable domain includes a LC variable region as set forth in SEQ ID NO:14. In some embodiments, the heavy chain variable domain includes a HC variable region as set forth in SEQ ID NO:13. In some embodiments, the light chain variable domain includes a LC variable region as set forth in SEQ ID NO:14, and the heavy chain variable domain includes a HC variable region as set forth in SEQ ID NO:13.Docket No.062822-501001WO

[0131] In some embodiments, the light chain variable domain includes a LC variable region that is more than 95%, 96%, 97%, 98%, or 99% idential to the amino acid sequence as set forth in SEQ ID NO:14. In some embodiments, the heavy chain variable domain includes a HC variable region that is more than 95%, 96%, 97%, 98%, or 99% idential to the amino acid sequence as set forth in SEQ ID NO:13.

[0132] In some embodiments, the light chain variable domain includes a LC as set forth in SEQ ID NO:10. In some embodiments, the light chain variable domain includes a LC as set forth in SEQ ID NO:6. In some embodiments, the heavy chain variable domain includes a HC as set forth in SEQ ID NO:9. In some embodiments, the heavy chain variable domain includes a HC as set forth in SEQ ID NO:7. In some embodiments, the heavy chain variable domain includes a HC as set forth in SEQ ID NO:8. In some embodiments, the heavy chain variable domain includes a HC as set forth in SEQ ID NO:21.

[0133] In some embodiments, the light chain variable domain includes a LC that is 95%, 96%, 97%, 98%, or 99% idential to the amino acid sequence as set forth in SEQ ID NO:10. In some embodiments, the light chain variable domain includes a LC that is 95%, 96%, 97%, 98%, or 99% idential to the amino acid sequence as set forth in SEQ ID NO:6. In some embodiments, the heavy chain variable domain includes a HC that is 95%, 96%, 97%, 98%, or 99% idential to the amino acid sequence as set forth in SEQ ID NO:9. In some embodiments, the heavy chain variable domain includes a HC that is 95%, 96%, 97%, 98%, or 99% idential to the amino acid sequence as set forth in SEQ ID NO:7. In some embodiments, the heavy chain variable domain includes a HC that is 95%, 96%, 97%, 98%, or 99% idential to the amino acid sequence as set forth in SEQ ID NO:8. In some embodiments, the heavy chain variable domain includes a HC that is 95%, 96%, 97%, 98%, or 99% idential to the amino acid sequence as set forth in SEQ ID NO:21.

[0134] In one embodiment, the antibody is nivolumab. Nivolumab (also known as "OPDIVO ®"; BMS-936558; formerly designated 5C4, BMS-936558, MDX-1106, or ONO-4538) is a fully human IgG4 (S228P) PD-1 immune checkpoint inhibitor antibody that selectively prevents interaction with PD-1 ligands (PD-L1 and PD-L2), thereby blocking the down-regulation of antitumor T-cell functions (U.S. Patent No.8,008,449; Wang et al, 2014 Cancer Immunol Res.2(9):846-56). In another embodiment, the anti-PD-1 antibody or fragment thereof cross-competes with nivolumab. In other embodiments, the anti- PD-1 antibody or fragment thereof binds to the same epitope as nivolumab. In certain embodiments, the anti-PD-1 antibody has the same CDRs as nivolumab.

[0135] In some embodiments, the antibody is Cemiplimab. Cemiplimab (also known as Libtayo® ) is a PD-1 immune checkpoint inhibitor antibody for the treatment of squamous cell skin cancer.Docket No.062822-501001WO

[0136] Anti-human-PD-1 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the invention can be generated using methods well known in the art. Alternatively, art recognized anti- PD-1 antibodies can be used. For example, monoclonal antibodies 5C4 (referred to herein as Nivolumab or BMS-936558), 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4, described in WO 2006 / 121168, the teachings of which are hereby incorporated by reference, can be used. Other known PD-1 antibodies include lambrolizumab (MK-3475) described in WO 2008 / 156712, and AMP-514 described in WO 2012 / 145493, the teachings of which are hereby incorporated by reference. Further known anti-PD-1 antibodies and other PD-1 inhibitors include those described in WO 2009 / 014708, WO 03 / 099196, WO 2009 / 114335 and WO 2011 / 161699, the teachings of which are hereby incorporated by reference. In one embodiment, the anti-PD-1 antibody is REGN2810. In one embodiment, the anti-PD-1 antibody is PDR001. Another known anti-PD-1 antibody is pidilizumab (CT-011). Antibodies or antigen binding fragments thereof that compete with any of these antibodies or inhibitors for binding to PD-1 also can be used.

[0137] In some embodiments, a sequence such as a light chain, heavy chain, VL, or VH sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence herein such as a light chain, heavy chain, VL, or VH sequence.

[0138] In some embodiments, the antibody has an increased serum half-life in a mammal compared to any antibody produced by expressing polypeptides having an endoplasmic reticulum retention signal, produced in plants genetically modified to generate human-like glycosylation patterns, or produced in a mammalian expression system.

[0139] In some embodiments, the antibody has an increased serum half-life in a mammal compared to an antibody produced in a mammalian expression system. In some embodiments, the antibody has more than 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% increased serum half-life in a mammal compared to an antibody produced in a mammalian expression system. In some embodiments, the antibody has more than double the serum half-life in a mammal compared to an antibody produced in a mammalian expression system.

[0140] In another aspect a composition comprising a plurality of antibodies of the disclosure is provided. In some embodiments, more than 80%, 85%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the plurality of antibodies comprise a GlcNAc2Man3GlcNAc2 (GnGn) glycosylation and lack β1,2-xylose and / or α1,3-fucose. In some embodiments, more than 80%, of the plurality of antibodies comprise a GlcNAc2Man3GlcNAc2 (GnGn) glycosylation and lack β1,2-xylose and / or α1,3-fucose. In some embodiments, more than 90% of the plurality of antibodies comprise a GlcNAc2Man3GlcNAc2 (GnGn) glycosylation and lack β1,2-xylose and / or α1,3-fucose. In some embodiments, more than 95% of the plurality of antibodies comprise a GlcNAc2Man3GlcNAc2 (GnGn) glycosylation and lack β1,2-Docket No.062822-501001WO xylose and / or α1,3-fucose. In some embodiments, more than 99% of the plurality of antibodies comprise a GlcNAc2Man3GlcNAc2 (GnGn) glycosylation and lack β1,2-xylose and / or α1,3-fucose. The percentage of glycosylation can be determined for example by comparing the ratio of the glycosylated peak in a mass spectrometry chromatogram, such as the GnGn-glycosylated antibody, to the non-glycosylated antibody. This provides a measure of the percentage of glycosylation, for example GnGn glycosylation of an antibody at a glycosylation site relative to the non-glycosylated antibody at the same site. III. METHODS OF MAKING ANTIBODIES

[0141] Provided herein are, inter alia, methods of making the antibodies and antibody compositions of the dislcosure.

[0142] In an aspect, a method of making an antibody in plant is provided, the method comprising expressing in a plant one or more antibody polypeptide sequences that lack an endoplasmic reticulum retention signal. Some embodiments relate to a plant comprising an expression vector. The expression vector may include a promoter sequence (such as a plant-specific promoter or a constitutive promoter) operably linked to one or more antibody sequences (such as a light chain sequence and / or a heavy chain sequence).

[0143] Antibodies can be produced in transgenic plants, as an inexpensive production alternative to existing mammalian systems. For example, the transgenic plant may be a tobacco plant, e.g., Nicotiania benthamiana or Nicotiana tabaccum. The transgenic plant may be a Nicotiania plant. The antibodies can be purified from plant material, for example from the plant leaves. Stable transformation of the plants can be achieved through the use of Agrobacterium tumefaciens or particle bombardment. For example, nucleic acid expression vectors containing at least the heavy and light chain sequences are expressed in bacterial cultures, i.e., A. tumefaciens strains BLA4404 or GV3101, via transformation. Infiltration of the plants can be accomplished via injection. Soluble leaf extracts can be prepared by grinding leaf tissue in a mortar and by centrifugation. Isolation and purification of the antibodies can be readily be performed by many of the methods known to the skilled artisan in the art. Other methods for antibody production in plants are described in, for example, Fischer et al., Vaccine (2003), 21:820-5; and Ko et al, Current Topics in Microbiology and Immunology (2009), 332, pp.55-78.

[0144] The disclosure further provides any cell or plant comprising a vector that encodes the antibody of the disclosure, or produces the antibody of the disclosure. In some embodiments, the plant is Nicotiana benthamiana. In some embodiments, the plant is not genetically engineered to alter glycosylation of the antibody. In some embodiments, the plant is genetically engineered to alter glycosylation of the antibody.Docket No.062822-501001WO In some embodiments, the plant is a glycoengineered plant. In some embodiments, the plant is a glycoengineered Nicotiana benthamiana.

[0145] In some embodiments, the plant is a wild-type plant. In some embodiments, the plant is a wild- type Nicotiana benthamiana. In some embodiments, the plant is a glycoengineered (ΔXF) Nicotiana benthamiana plant. Glycoengineered ΔXF Nicotiana benthamiana plants are known in the art, for example at Strasser et al., Plant Biotechnology Journal (2008) 6(4): 392-402. ΔXF plants can have reduced xylosyl or fucosyl transferase enzymatic activities.

[0146] In some embodiments, the plant is ΔXF Nicotiana benthamiana.

[0147] In some embodiments, the plant is transformed with a bacterial vector comprising a nucleic acid encoding for the antibody. In some embodiments, the bacterial vector is Agrobacterium tumefaciens.

[0148] In some embodiments, the antibody is purified from the plant material. In some embodiments, the antibody is purified from the leaves of the plant. IV. PHARMACEUTICAL COMPOSITIONS

[0149] The compositions provided herein include pharmaceutical compositions including therapeutically effective amount of the anti PD-1 antibodies and antibody compositions provided herein including embodiments thereof. Thus, in an aspect is provided a pharmaceutical composition including a therapeutically effective amount of an antibody as provided herein including embodiments thereof and a pharmaceutically acceptable excipient.

[0150] In embodiments, the pharmaceutical composition further includes a therapeutically effective amount of a Programmed Death 1 (PD-1) inhibitor. In embodiments, the PD-1 inhibitor is Nivolumab, Pembrolizumab, or Cemiplimab. In embodiments, the PD-1 inhibitor is Nivolumab. In embodiments, the PD-1 inhibitor is Pembrolizumab. In embodiments, the PD-1 inhibitor is Cemiplimab. In embodiments, the therapeutically effective amount of the antibody and the therapeutically effective amount of the PD-1 inhibitor is a combined synergistic amount.

[0151] A therapeutically effective amount as provided herein refers to an amount effective to achieve its intended purpose. The actual amount effective for a particular application will depend, inter alia, on the condition being treated. When administered in methods to treat a disease, the pharmaceutical compositions described herein will contain an amount of active antibody effective to achieve the desired result, e.g., modulating the activity of a target molecule (e.g., PD-1), and / or reducing, eliminating, or slowing the progression of disease symptoms (e.g., cancer). Determination of a therapeutically effective amount of an antibody provided herein is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure herein.Docket No.062822-501001WO

[0152] Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, or acetate at a pH typically of 5.0 to 8.0, most often 6.0 to 7.0; salts such as sodium chloride, potassium chloride, etc. to make isotonic; antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers such as polysorbate 80, amino acids such as glycine, carbohydrates, chelating agents, sugars, and other standard ingredients known to those skilled in the art (Remington’s Pharmaceutical Science 16thedition, Osol, A. Ed.1980). The mAb is typically present at a concentration of 0.1 - 100 mg / ml, e.g., 1 - 10 mg / ml or 10 - 50 mg / ml, for example 5, 10, 20, 30, 40, 50 or 60 mg / ml.

[0153] A pharmaceutical composition including a PD-1 antibody as described herein can be administered by a variety of methods known in the art. The route and / or mode of administration vary depending upon the desired results. In embodiments, administration is intravenous, intramuscular, intraperitoneal, or subcutaneous, or administered proximal to the site of the target. Pharmaceutically acceptable excipients can be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).

[0154] Pharmaceutical compositions of the antibody can be prepared in accordance with methods well known and routinely practiced in the art. See, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20thed., 2000; and Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. Pharmaceutical compositions are preferably manufactured under GMP conditions. Typically, a therapeutically effective dose or efficacious dose of the antibody is employed in the pharmaceutical compositions of the invention. The antibodies provided can be formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It may be advantageous to formulate the antibodies in combination with other therapies or agents. It can be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of antibody calculated to produce the desired therapeutic effect in association with the required pharmaceutical excipient.

[0155] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level depends upon a variety of pharmacokinetic factorsDocket No.062822-501001WO including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular antibody being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors.

[0156] The antibody provided herein can be administered on multiple occasions. Intervals between single dosages can be weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of the antibody in the patient. In some methods, dosage is adjusted to achieve a plasma antibody concentration of 1–1000 ^g / ml and in some methods 25–300 ^g / ml. Alternatively, antibody can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the subject. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some subjects continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, the subject can be administered a prophylactic regime. V. METHODS OF TREATMENT

[0157] The antibodies and pharmaceutical compositions provided herein, including embodiments thereof, are contemplated as providing effective treatments for diseases associated with PD-1 function and / or of inhibiting PD-1 function such as cancer. In an aspect, a method of treating or preventing a condition associated with PD-1 function and / or of inhibiting PD-1 function in vivo is provided, comprising administering to a subject an antibody of the disclosure.

[0158] In an aspect is provided a method of treating cancer in a subject in need thereof, the method including administering to a subject a therapeutically effective amount of an antibody as provided herein including embodiments thereof, thereby treating cancer in the subject.

[0159] In embodiments, the antibody exhibits increased half-life in the subject compared to any antibody produced by expressing polypeptides having an endoplasmic reticulum retention signal or produced in a mammalian expression system.

[0160] In embodiments, the antibody exhibits increased half-life in the subject compared to any antibody produced by expressing polypeptides having an endoplasmic reticulum retention signal,Docket No.062822-501001WO produced in plants genetically modified to generate human-like glycosylation patterns, or produced in a mammalian expression system.

[0161] In embodiments, the serum concentration of antibody in the subject is increased at 4 hours, 24 hours, or 48 hours compared to any antibody produced by expressing polypeptides having an endoplasmic reticulum retention signal, or produced in a mammalian expression system.

[0162] In embodiments, the serum concentration of antibody in the subject is increased at 4 hours, 24 hours, 48 hours compared to any antibody produced by expressing polypeptides having an endoplasmic reticulum retention signal, produced in plants genetically modified to generate human-like glycosylation patterns, or produced in a mammalian expression system. EXAMPLES

[0163] These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein. Example 1. Production, Purification, and Activity of a glycosylation modified anti PD-1 antibody

[0164] In eukaryotes, signal sequences are N-terminal extensions of newly synthesized secretory and membrane proteins. Signal sequences can have important roles in modulating protein biogenesis by containing information that specifies the choice of targeting pathway, the efficiency of translocation, and even post-translational functions. Incorporating protein glycan moiety remodeling which contains the addition of signal peptide sequence into the plant expression system demonstrates the production of various glycoforms that might mimic the human-like N-glycosylation with proper folding to maintain the structural integrity and functioning. Plants are excellent hosts with litheness to undergo genome manipulation to get rid of plant specific glycans and their antigenicity in mammalian subjects. Likewise, the impact of the different glycoprofiles of an antibody on the interaction with its receptor along with the biodistribution of each antibody variant in the blood stream defines the antibodies efficacy and functionality.

[0165] To determine the impact of different glycosylation patterns on the efficacy, functionality and half life of antibodies in mammalian subject, several antibodies and expression hosts were tested. Antibody Design and Cloning

[0166] Briefly, various forms of pembrolizumab (anti PD-1) antibody were constructed containing a signal peptide and with or without SEKDEL sequence which aids in retrograde transport and was produced either in wild-type or glycoengineered Nicotiana benthamiana. The constructed clones were confirmed, produced in the hosts, purified by affinity chromatography and additionally used for in vivo study to assess the biodistribution in the blood stream.Docket No.062822-501001WO

[0167] Antibody constructs prepared for this study are listed in Table 2. Table 2 Amino acid sequences of exemplary anti PD-1 antibodies Antibody Alternative Name Target SEQ ID NOs: S-BPM-001-WT / Pembro-WT PD-1 7, 6 S-BPM-001-KD-WT Pembro-KD PD-1 8, 6 S-BPM-001-ΔXF Pembro -XF PD-1 7, 6 S-BPM-001-N297A-WT Pembro-NG PD-1 21, 6 Keytruda® Keytruda PD-1 9, 10

[0168] Briefly, the gene fragments encoding Pembrolizumab or BPM-001 (Drug bank accession number DB09037) heavy chain (HC) and light chain (LC) were codon-optimized in silico for the expression in N. benthamiana. Expression of antibodies in N. benthamiana has been described elsewhere (e.g., Phakam et al., Front. Plant Sci., Sec. Plant Biotechnology (2021) 12). The plant-optimized codon sequences were synthesized. The variable domains of BPM-001 were fused separately to the constant domains of human IgG4 gamma chain for HC and human IgG4 kappa chain for LC. Prior to ligation, the variable and constant domains of the antibody as well as the pBaiYa / pBY2eK vector were digested using restriction enzymes (Table 3). Both the full-length HC and LC sequences were flanked with a murine leader sequence at the N-terminus and with or without Ser-Glu- Lys-Asp-Glu-Leu (SEKDEL) sequence at the C-terminus of HC. In addition, an amino acid substitution was incorporated into the N-glycan site at Asn 297 (N297A) by overlapping PCR method to generate a non-glycosylated BPM-001. The primers used to introduce N297A mutation are listed in Table 4. A visual representation of the expected antibody structures and N-glycan structures of BPM-001 glycovariants is provided in FIGs.1-4. The Pembrolizumab HC and LC gene fragments were double digested, purified and cloned into plant expression vector pBaiYa. The LC with signal peptide and HC with or without SEKDEL in expression vectors were transformed into Escherichia coli DH10B by heat chock method and colonies were screened by polymerase chain reaction (PCR) using vector specific primers (Table 4). Positive clones were further cultivated in kanamycin-selective broth and plasmid DNAs were extracted by DNA-spin™ plasmid DNA purification kit. Purified plasmids containing the HC and LC of BPM-001 were transformed into Agrobacterium tumefaciens GV3101 by electroporation and then cells were spread onto antibiotic-selective agar media supplemented with kanamycin, rifampicin, and gentamicin. Colonies were screened by PCR and gene fragments were confirmed by running on an agarose gel electrophoresis. Table 3. List of restriction enzymes used for digestion of BPM-001 variable and constant domains. Gene Restriction enzyme / s S-BPM-001-VL XbaI / AflIIDocket No.062822-501001WO IgG4-CL AflII / SacI S-BPM-001-VH XbaI / BmtI IgG4-CH BmtI / SacI pBaiYa / pBY2eK XbaI / SacI Table 4. Primers used for non-glycosylated BPM-001 heavy chain Primer name 5’ – 3’ Sequencing IgG4-N297A_F primer CGTGAGGAACAGTTCGCAAGCACCTACAGGGTTG IgG4-N297A_R primer CAACCCTGTAGGTGCTTGCGAACTGTTCCTCACG Table 5. Primers used for colony PCR Primer name 5’ – 3’ Sequencing F primer GGAGAGGACCTCGAGAAAC R primer GCTTTGCATTCTTGACATC

[0169] The variable and constant domains of BPM-001 as well as the pBaiYa vector was enzyme digested prior ligation and cloning (FIG.5). The plant expression vector harboring BPM-001 LC and HC variable regions in humanized IgG4 with signal peptide and with or without KDEL have been cloned by PCR. The cloned fragments were transformed into E. coli strain DH10B (FIG.5). Further, the recombinant clones screened for the target gene fragments were used for plasmid extraction. The isolated plasmid was confirmed by restriction enzyme digestion and positive clones were then transformed into Agrobacterium tumefaciens strain GV3101 (FIG.6). Based on the results, LC and HC gene inserts were detected at expected base pair (bp) sizes of 750 bp and 1,500 bp, respectively. Following transformation, selected colonies were amplified and showed the presence of LC and HC genes, as predicted. Nucleotide sequences of synthesized LC and HC were verified by Sanger sequencing. Amino acid sequences of BPM-001 antibodies are shown in Tables 6-10. Table 6 Detailed description of the S-BPM-001-LC amino acidAmino acid sequenceSEQ IDsequenceNO:Description and Accession No. Signal peptide MGWSCIILFLVATATGVHS 11 Accession No. P01750Docket No.062822-501001WO Part of restriction DVQLLE 12 enzyme sequence Light Chain EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRL 10 Sequence LIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTF Accession No. GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW DB09037 KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC Table 7 Detailed description of the S-BPM-001- HC amino acid Amino acid sequence SEQ ID NO: sequence Description and Accession No. Signal peptide MGWSCIILFLVATATGVHS 11 Accession No. P01750 Part of restriction DVQLLE 12 enzyme sequence Heavy Chain QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPG 9 Sequence QGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKS Accession No. LQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPS DB09037 VFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVD KRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHN HYTQKSLSLSLGK Table 8 Detailed description of the S-BPM-001- HC-KD amino acid Amino acid sequence Seq ID NO: sequence Description and Accession No. Signal peptide MGWSCIILFLVATATGVHS Accession No. 11 P01750 Part of restriction DVQLLE enzyme sequence 12 Heavy Chain QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPG Sequence QGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKS Accession No. LQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPS DB09037 VFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH 9 TFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVD KRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQDocket No.062822-501001WO VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHN HYTQKSLSLSLGK Retention signal SEKDEL sequence Table 9 Detailed description of the S-BPM-001- Amino acid sequence Seq ID NO: N297A HC-KD amino acid sequence Signal peptide MGWSCIILFLVATATGVHS Accession No. 11 P01750 Part of restrictionDVQLLEenzyme sequence12Heavy Chain QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQA PGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMEL Sequence KSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS Accession No. ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA DB09037 LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS NTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR 21 TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFAS TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM HEALHNHYTQKSLSLSLGK Retention signal SEKDEL sequence Table 10 Detailed description of the S-BPM-001- Amino acid sequence Seq ID NO: N297A HC-KD amino acid sequence Signal peptide MGWSCIILFLVATATGVHS 11 Accession No. P01750 Part of restriction DVQLLE 12 enzyme sequence Heavy Chain QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQA 21 PGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMEL Sequence KSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS Accession No. ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA DB09037 LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS NTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFAS TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGDocket No.062822-501001WO QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM HEALHNHYTQKSLSLSLGK Protein Expression

[0170] The Agrobacterium cultures of BPM-001 HC and LC were used for agroinfiltration into wild- type and glycoengineered N. benthamiana plant for recombinant antibody production. The different glycovariants were infiltrated in wild-type (WT) and glycoengineered (ΔXF) N. benthamiana plants. After infiltration, the plants were incubated under controlled environment for 3-5 days. Leaves were then harvested, extracted in 1X PBS, centrifuged.

[0171] The leaf extracts were purified by protein A chromatography and the purified glycovariant proteins were confirmed by SDS-PAGE and Western blot (FIG.7). For the SDS-PAGE analysis, the bands (2 μg) were visualized by One-Step Blue® staining (Biotium, USA). For western blot, approximately 0.05–0.5 μg of antibodies were transferred onto nitrocellulose membrane (Bio-Rad, CA, United States). The membrane was blocked with 5% skim milk in PBS and then washed with PBST. Proteins were detected either with HRP-conjugated anti-human gamma antibody (SouthernBiotech, USA) or HRP-conjugated anti-human kappa antibody (SouthernBiotech, USA) diluted (1:5,000) in PBS. The membranes were washed with PBST, developed using enhanced chemiluminescence (ECL) plus detection reagent (Abcam, United Kingdom), and recorded in a medical X-ray green (MXG) film (Carestream, GA, United States). The results show, that BPM-001 glycovariants were successfully assembled at approximately 150 kDa under non-reducing condition with 2 HC and 2 LC proteins, each chain at approximately 50 kDa and 25 kDa under reducing condition. Western blot results also confirmed the presence of HC as detected by anti-Gamma antibody and LC as detected by anti-Kappa antibody at expected protein sizes.

[0172] The results show that mutated BPM-001-N297A variants were successfully expressed and purified in WT N. benthamiana plants. The results show that the antibody assembled at approximately 150 kDa under non-reducing condition (FIG.9).

[0173] The purified BPM-001 glycovariants were also tested for binding efficacy to human PD-1 target using functional ELISA. Commercial Keytruda® was used as a positive control. . A 96-well plate was coated with 2 μg / ml of recombinant human PD-1 His tag protein (8986-PD, R&D Systems, MN, United States) at 4°C for overnight. Then, the plate was washed and blocked with PBST. Each BPM-001 antibody and commercial Keytruda® was serially diluted, added on the coated plate, and incubated for 2 h at 37 °C. Bound BPM-001 glycovariants were captured by goat anti-human Kappa-HRP (SouthernBiotech, Alabama, USA) at 1:3,000 in 1×PBS, and the enzyme activity was determined by TMB one solution substrate (Promega, Wisconsin, USA). The reaction was quenched with 1M H2SO4and the absorbance was measured at 450 nm using a spectrophotometer.Docket No.062822-501001WO

[0174] The results show that all plant-produced BPM-001 antibodies could bind specifically to PD-1 protein and had comparable binding to Keytruda® (FIG.10 and Table 11). Pembrolizumab produced in WT plants (S-BPM-001-WT) binds to human PD-1 with EC50 of 0.0383 µg / mL. Pembrolizumab containing SEKDEL peptide and produced in WT plants (S-BPM-001-KD-WT) binds to human PD-1 with EC50 of 0.0312 µg / mL. Pembrolizumab produced in ΔXF plants (S-BPM-001-ΔXF) binds to human PD-1 with EC50 of 0.0288 µg / mL. Pembrolizumab containing N297A mutation and produced in WT plants (S-BPM-001-N297A-WT) binds to human PD-1 with EC50 of 0.0436 µg / mL. Table 11. EC50 values of BPM-001 glycovariants for target binding Antibody Target EC50(µg / mL) R2S-BPM-001-WT PD-1 0.0383070 0.997568 S-BPM-001-KD-WT PD-1 0.0311551 0.992894 S-BPM-001-ΔXF PD-1 0.0288485 0.993753 S-BPM-001-N297A-WT PD-1 0.0435647 0.997669 Keytruda® PD-1 0.0200727 0.999256

[0175] The purified antibodies were subjected for further analysis of the glycosylation patterns on the polypeptide to LC MS.

[0176] LC-MS analysis revealed that the subunit mass of Pembrolizumab antibodies ranged from 50 to 52 kDa (FIG.11). The observed mass increase compared to the theoretical mass of 50 kDa is likely attributable to post-translational modifications, with N-glycosylation at amino acid residue contributing approximately 1.2–1.8 kDa of glycan unit to the molecular mass. The MS data identified distinct glycan patterns at the N297 site (amino acid numbering with respect to the conserved glycosylation site at amino acid N297 in the CH2 domain of IgG1). The results show, that the peptide sequence EEQFNSTYR (comprising N297 of the glycosylation motif NxT) was found attached with different plant-derived glycans: GlcNAc2Man3GlcNAc2 (GnGn) for S-BPM-001-ΔXF, the xylosylated and core α1,3- fucosylated N-glycan GnGnXF for S-BPM-001-WT, and Man7-9GlcNAc2 for S-BPM-001-KD-WT. As expected, no glycan species were detected for the antibody variant comprising the N297A mutation (S- BPM-001-N297A-WT). Keytruda / pembrolizumab showed G0F (NGA2F) glycosylation pattern. Example 2. Biodistribution of glycosylation modified anti PD-1 antibody in vivo

[0177] The purified proteins were also used for an initial biodistribution study in mice in a short time frame at 3 mg / kg.

[0178] The purified proteins from Example 1 were injected into 43-day-old mice intravenously at a dosing regimen of 3 mg / kg for biodistribution analysis.Docket No.062822-501001WO

[0179] Briefly, 6-week-old (day 43) mice were grouped into 4 groups (n=1 mouse / group). The dosing of respective BPM-001 glycovariants along with commercial Keytruda® was administered single time at a dosing concentration of 3 mg / kg intravenously. Peripheral blood was collected at 4 h, 24 h and 48 h post administration to assess the concentration of the drug and its clearance rates by ELISA. Table 12 shows the experimental plan for the biodistribution analysis. Table 12. Study plan for pembrolizumab (BPM-001) distribution in mice Dosage Blood collection Group No. Treatment ROA Frequency (mg / kg) 4h 24h 48h 1 1 S-BPM-001-WT 3 i.v. single dose √ √ √ 2 1 S-BPM-001-KD-WT 3 i.v. single dose √ √ √ 3 1 S-BPM-001-ΔXF 3 i.v. single dose √ √ √ 4 1 Keytruda® 3 i.v. single dose √ √ √ Notes: No., animal number; ROA, route of administration; i.v., intravenous injection; h, hour. The amount of protein used is 120 µg / mice with body weight of 40 g.

[0180] After the administration, peripheral blood was collected at 4 h, 24 h and 48 h to assess the concentration and distribution of BPM-001 in the animal body. The collected blood samples were used in the PD-1 binding assay to estimate the concentration of pembrolizumab drug.

[0181] The PD-1 Elisa binding activity of different glycovariant plant-produced BPM-001 antibodies in the blood drawn samples of mice were determined by ELISA. Briefly, the ELISA plate was coated with 25 μl / well of recombinant human PD-1 His tag protein at 2 μg / ml (8986-PD, R&D Systems, MN, United States) at 4°C for overnight. The plate was washed and blocked with PBST. Each BPM-001 glycovariant was spiked into naive serum with different concentration for standard curve. The pembrolizumab in naive and serum samples were added and the plate was incubated at 37°C for 2 h. Then, the HRP-conjugated anti-human kappa antibody (SouthernBiotech, USA) diluted (1:10,000) in PBS was added and incubated at 37°C for 1 h. The plate was washed and developed with 25 μl / well of TMB One Solution (Promega, USA). The reaction was stopped by adding 25 μl / well of 1 M H2SO4 and the absorbance was measured at 450 nm using a spectrophotometer. Each BPM-001 glycovariant was used as individual standard.

[0182] S-BPM-001-WT, which is the signal peptide containing pembrolizumab expressed in wild type plants, showed the maximum distribution rate with a concentration of 49.8 ug / mL at 4 h and 31.9 ug / mL at 48 h. Commercial Keytruda® also showed similar distribution rate of 36 ug / mL and 27.7 ug / mL respectively. Meanwhile, S-BPM-001-KD-WT, which is the signal peptide and SEKDEL containing pembrolizumab produced in wild-type, showed a concentration of 28.6 ug / mL at 4 h and 16.1 ug / mL atDocket No.062822-501001WO 48 h. The purified antibody S-BPM-001-ΔXF with signal peptide produced in glycoengineered plant also showed lower distribution with faster clearance as shown in Table 13. Table 13. Distribution of glycovariant pembrolizumab proteins Protein 4hr (µg / ml) 24hr (µg / ml) 48hr (µg / ml) S-BPM-001-WT 49.8 34.4 31.9 S-BPM-001-KD-WT 28.6 24.3 16.1 S-BPM-001-ΔXF 24.5 17.1 15.9 Keytruda® 36.0 30.8 27.7

[0183] The results show, that the plant-derived pembrolizumab containing a signal peptide and produced in wild-type N. benthamiana (S-BPM-001-WT) showed the maximum biodistribution in mice within the first 4h, accumulating to a concentration of 49.8 ug / mL with slower clearance rates until 48h with a concentration of 31.9 ug / mL. Example 3. Biodistribution of glycosylation modified anti PD-1 antibody in vivo

[0184] This example describes the determination of the biodistribution of plant expressed and purified antibodies in comparison with commercial mammalian cell produced antibody in a longer time frame at 5mg / kg.

[0185] Briefly, plant-produced purified antibodies were evaluated for biodistribution study in mice.6- week-old mice were grouped into 5 groups (n=8 mice / group). The dosing of respective BPM-001 glycovariants along with commercial Keytruda® was administered at a single time at a dosing concentration of 5 mg / kg intravenously. Peripheral blood was collected at 0 h (pre-dose), 0.5 h, 1 h, 2 h, 8 h, 24 h, 48 h, 72 h, 96 h, and 168 h post administration to assess the concentration of the antibody and its clearance rates by ELISA. Table 14 shows the experimental plan for the biodistribution analysis.

[0186] Purified proteins were injected intravenously into six-week-old mice at a dose of 5 mg / kg for biodistribution analysis (FIG.14). Table 14. Study plan for pembrolizumab (BPM-001) distribution in mice Group AntibodyDosageROAMiceQuantityTimepoint of blood collection (mpk) 0 h (pre-dose), 1h, 8h, 48h, 96h G1 Keytruda® 5 i.v.44 0.5h, 2h, 24h, 72h, 168h 4 0 h (pre-dose), 1h, 8h, 48h, 96h G2 S-BPM-001-WT 5 i.v.40.5h, 2h, 24h, 72h, 168h 4 0 h (pre-dose), 1h, 8h, 48h, 96h G3 S-BPM-001-ΔXF 5 i.v.40.5h, 2h, 24h, 72h, 168hDocket No.062822-501001WO G4S-BPM-001-KD-40 h (pre-dose), 1h, 8h, 48h, 96h WT 5 i.v.40.5h, 2h, 24h, 72h, 168h G5S-BPM-001-5 i.v. 40 h (pre-dose), 1h, 8h, 48h, 96h N297A-WT 40.5h, 2h, 24h, 72h, 168h

[0187] The concentration of various glycovariant plant-produced BPM-001 antibodies in mouse blood samples was determined using either indirect or sandwich enzyme-linked immunosorbent assay (ELISA). An ELISA plate was coated with 50 μL per well of recombinant human PD-1 His-tag protein (8986-PD, R&D Systems, MN, USA) or goat polyclonal antibody to human IgG1 (Cat No: ab97221, Abcam, United Kingdom) at a concentration of 2 μg / mL and incubated at 4°C overnight. The plate was then washed with PBST and blocked with 3% BSA in PBS. To establish a standard curve, Keytruda, S-BPM-001-KD-WT glycovariant, or human IgG1 kappa isotype were spiked into naive serum at varying concentrations. A 2.5-fold serial dilution was prepared, starting from 0.4 µg / mL for Keytruda or S-BPM-001-KD-WT and 1.0 µg / mL for the human IgG standard. The standard calibration curve and serial dilutions of pembrolizumab glycovariants in serum samples were added to the plate and incubated at 37°C for 2 h. For detection, HRP-conjugated anti-human kappa antibody (SouthernBiotech, USA) diluted 1:7,000 or anti-human IgG4 antibody (SouthernBiotech, USA) diluted 1:50,000 in PBS were added to the wells and incubated at 37°C for 1 h. The plate was washed and developed with 50 μL per well of TMB One Solution (Promega, USA). The reaction was stopped by adding 50 μL per well of 1 M H₂SO₄, and the absorbance was measured at 450 nm using a spectrophotometer.

[0188] The serum concentrations of the glycosylation variants of BPM-001 over time are shown in FIG.13. The results show the half live of S-BPM-001-ΔXF in mice exhibited a longer half-life of 45.83 h in a single administration study (FIG.14), compared to the half-life of commercial Keytruda at 33.26 h. This indicates a improved PK profile for plant-produced mAbs lacking xylose and fucose residues compared to Keytruda antibody. In contrast, shorter half-lives of 26.70 h and 32.95 h were observed for S-BPM-001-WT and S-BPM-001-KD-WT, respectively, suggesting that plant-specific glycans and high- mannose type glycans contribute to a reduced distribution of plant produced Pembrolizumab in mice. In addition, S-BPM-001-N297A-WT (comprising no glycosylation) demonstrated a half-life of 34.27 h, comparable to that of Keytruda, indicating no significant impact on pharmacokinetics and a similar profile to the glycosylated antibodies. The comparison of half-lives between S-BPM-001-ΔXF and Keytruda across two ELISA detection methods showed consistent trends: 45.83 h versus 33.26 h (Indirect ELISA) and 36.08 h versus 28.86 h (Sandwich ELISA), respectively (FIG.15).

[0189] While the disclosure has been particularly shown and described with reference to specific embodiments (some of which are preferred embodiments), it should be understood by those having skillDocket No.062822-501001WO in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the present disclosure as disclosed herein. REFERENCES Leipold D, Prabhu S. Pharmacokinetic and Pharmacodynamic Considerations in the Design of Therapeutic Antibodies. Clin Transl Sci.2019 Mar;12(2):130-139. doi: 10.1111 / cts.12597. Epub 2018 Dec 27. PMID: 30414357; PMCID: PMC6440574. Liming Liu, Pharmacokinetics of monoclonal antibodies and Fc-fusion proteins, Protein & Cell, Volume 9, Issue 1, January 2018, Pages 15–32. Strasser R. Plant protein glycosylation. Glycobiology.2016 Sep;26(9):926-939. doi: 10.1093 / glycob / cww023. Epub 2016 Feb 23. PMID: 26911286; PMCID: PMC5045529. Strasser R, Seifert G, Doblin MS, Johnson KL, Ruprecht C, Pfrengle F, Bacic A and Estevez JM (2021) Cracking the “Sugar Code”: A Snapshot of N- and O-Glycosylation Pathways and Functions in Plants Cells. Front. Plant Sci.12:640919. doi: 10.3389 / fpls.2021.640919 Gomord, V., Fitchette, A.-C., Menu-Bouaouiche, L., Saint-Jore-Dupas, C., Plasson, C., Michaud, D. and Faye, L. (2010), Plant-specific glycosylation patterns in the context of therapeutic protein production. Plant Biotechnology Journal, 8: 564-587. INCORPORATION BY REFERENCE

[0190] The entire disclosure of each of the patent and scientific documents referred to herein is incorporated by reference for all purposes. EQUIVALENTS

[0191] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. The scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.Docket No.062822-501001WO SEQUENCE LISTING SEQ Name Sequence ID NO: 1 S-BPM- ACACAAGTATAGAAATAACGCATTTCCAATTCTTTGAAATTTCTGCAACATCTAGAAC 001-LC AATGGGCTGGTCCTGCATCATCCTGTTCCTTGTTGCTACTGCTACCGGCGTTCACTCTG ATGTTCAACTTCTCGAGGAAATTGTGCTTACCCAGTCTCCTGCTACCTTGTCTTTGTCT CCTGGTGAGAGGGCTACTTTGTCTTGCAGAGCTTCTAAGGGCGTGAGCACCTCTGGTT ACTCTTACCTTCATTGGTATCAGCAGAAGCCAGGTCAGGCTCCTAGGCTTCTTATCTA CCTTGCCAGCTACCTTGAGTCTGGTGTTCCTGCTAGGTTCAGCGGTTCTGGTTCTGGT ACTGATTTCACCCTGACCATCAGCTCACTTGAGCCTGAGGATTTCGCTGTGTACTACT GCCAGCACTCTAGGGATCTTCCACTTACTTTCGGTGGTGGTACCAAGGTTGAGATTAA GAGGACTGTTGCTGCCCCGTCCGTGTTCATTTTTCCACCATCTGATGAGCAGCTGAAG TCCGGTACTGCTTCTGTTGTGTGCCTGCTGAACAACTTCTACCCTAGGGAAGCTAAGG TGCAGTGGAAGGTTGACAACGCACTTCAGAGCGGTAACAGCCAAGAGTCTGTTACCG AGCAGGACAGCAAGGACTCTACCTACTCTCTTAGCTCTACCCTGACCTTGAGCAAGG CCGATTACGAGAAGCACAAGGTGTACGCTTGCGAGGTTACACACCAGGGACTTTCTT CACCTGTGACCAAGTCTTTCAACAGGGGAGAGTGCTGAGAGCTCGAAGTGACATCAC AAAGTTGAAGGTAATAAAGCCAAATTAATTAAGACATTTTCATAATGATGTCAGAAA GAGGAAGAGAAAA 2 ACCCAATATAAGAAATAACGCATTTCCAATTCTTTGAAATTTCTGCAACATCTAGAAC AATGGGCTGGTCCTGCATCATCCTGTTCCTTGTTGCTACTGCTACCGGCGTTCACTCTG ATGTTCAACTTCTCGAGCAGGTTCAGCTTGTTCAGTCTGGTGTCGAGGTGAAGAAACC TGGCGCTTCTGTGAAAGTGAGCTGCAAGGCTTCTGGTTACACCTTCACCAACTACTAC ATGTACTGGGTGAGACAGGCTCCTGGTCAAGGTCTTGAATGGATGGGTGGTATCAAC CCTAGCAATGGTGGCACCAACTTCAACGAGAAGTTCAAGAACAGGGTGACCCTGACC ACCGATTCTTCTACTACTACCGCCTACATGGAACTGAAGTCCCTCCAGTTCGATGACA CCGCTGTTTACTATTGCGCTCGGAGGGATTACAGGTTCGATATGGGTTTCGATTACTG GGGTCAGGGTACCACCGTGACCGTTTCTTCTGCTAGCACCAAGGGACCTTCTGTGTTC CCTCTTGCTCCTTGCTCTAGGTCTACTTCTGAGTCTACTGCTGCTCTTGGTTGCCTGGT GAAGGATTACTTTCCTGAGCCTGTGACCGTGAGCTGGAATAGTGGTGCTCTTACTTCT GGCGTGCACACTTTCCCTGCTGTGCTTCAATCTAGCGGCCTGTACTCTCTTTCTTCTGT S-BPM- GGTGACTGTGCCGTCCAGCTCTCTTGGTACTAAGACCTACACTTGCAACGTGGACCAC 001-HC AAGCCGTCTAATACCAAGGTGGACAAGCGGGTCGAGTCTAAGTATGGTCCTCCTTGT For CCTCCATGTCCAGCTCCAGAATTTCTTGGTGGTCCTAGCGTGTTCTTGTTCCCGCCTAA GCCTAAGGATACCCTGATGATTTCTCGGACTCCTGAGGTTACCTGCGTTGTGGTTGAT GTGAGCCAAGAGGACCCAGAGGTTCAGTTCAACTGGTATGTGGATGGTGTTGAGGTG CACAACGCTAAGACTAAGCCTCGTGAGGAACAGTTCAACAGCACCTACAGGGTGTGT CTGTGCTTACCGTGCTTCATCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGG TGAGCAACAAGGGCCTGCCGTCCTCTATGAAAAAACATCTCTAAGGCTAAGGGCCAG CCTAAGAAACCGCAAGTTTACCCCTTCCGCCGAGCCAGAAAAAATGACCAAAAATCA GGGGACCCTTACTGCCCCCCGGGAAGGGTTTTTCCCCCCCACATTCTGTCGAGGGGA ATAAAGGGCCCCCCAAAAAAACTAAAAAAATCCCCCCCTTGGGGGGTTCAAGGGTTT TTTCCTTGCAAGGGGGGAACCGGAAAAAAAAAAAGAGACGGAGGGAAAATTTCCTC CTTTGTTCTTAGGGGCTCCCCACCACCCCCAAAATTTCCCCCCCCCGAGGAAAGGACA CAAGCCACCCAGGAGGAGAAAACAAAATAATTTTTTCTTG 3 GAAGCTTATTAATTTGGCTTTATTACCTTCAACTTTGTGATGTCACTTCGAGCTCTCAC TTTCCAAGGGACAGGGACAGAGACTTCTGGGTGTAGTGATTGTGCAGAGCCTCATGC ATAACAGAGCAGCTGAACACGTTACCCTCCTGCCATCTTGACTTATCCACGGTCAGCC S-BPM- TGCTGTACAGGAAGAATGAACCATCGGAATCCAGCACAGGAGGGGTAGTCTTGTAGT 001-HC TGTTCTCGGGCTGACCATTAGACTCCCACTCGACAGCAATGTCGCTAGGGTAAAATCC rev CTTCACGAGGCAGGTAAGGCTCACCTGATTCTTGGTCATCTCTTCTTGGCTCGGCGGA AGAGTGTAAACTTGCGGTTCTCTAGGCTGGCCCTTAGCCTTAGAGATGGTCTTTTCAA TAGAGGACGGCAGGCCCTTGTTGCTCACCTTGCACTTGTACTCTTTGCCGTTCAGCCADocket No.062822-501001WO ATCCTGATGAAGCACGGTAAGCACAGACACAACCCTGTAGGTGCTGTTGAACTGTTC CTCACGAGGCTTAGTCTTAGCGTTGTGCACCTCAACACCATCCACATACCAGTTGAAC TGAACCTCTGGGTCCTCTTGGCTCACATCAACCACAACGCAGGTAACCTCAGGAGTC CGAGAAATCATCAGGGTATCCTTAGGCTTAGGCGGGAACAAGAACACGCTAGGACC ACCAAGAAATTCTGGAGCTGGACATGGAGGACAAGGAGGACCATACTTAGACTCGA CCCGCTTGTCCACCTTGGTATTAGACGGCTTGTGGTCCACGTTGCAAGTGTAGGTCTT AGTACCAAGAGAGCTGGACGGCACAGTCACCACAGAAGAAAGAGAGTACAGGCCGC TAGATTGAAGCACAGCAGGGAAAGTGTGCACGCCAGAAGTAAGAGCACCACTATTC CAGCTCACGGTCACAGGCTCAGGAAAGTAATCCTTCACCAGGCAACCAAGAGCAGCA GTAGACTCAGAAGTAGACCTAGAGCAAGGAGCAAGAGGGAACACAGAAGGTCCCTT GGTGCTAGCAGAAGAAACGGTCACGGTGGTACCCTGACCCCAGTAATCGAAACCCAT ATCGAACCTGTAATCCCTCCGAGCGCAATAGTAAACAGCGGGGTCATCGAACTGGAG GGACTTCAGTTCCATGTAGGCGTAATAATAAAAAAATCGGTGGTCAGGGTCACCCTG TTCTTGAACTTTCCTTGAAATTGGGGGCCCCCCTTTGTAGGGGTTGGAAACCCCCCCT CCTTTAAAAACTTGGGCACGGGAGCCGTTTTCCCCCACAAATATGTAAAAAGTGGGG GGAGGGGGTAACAAAAACTTTTCCCCCTCTTTTCAAAAAAGCCGCGGTTTTTTTTTCC CTCTCCCACCCACAACAAAAAAAAAAAATAAAACCCTCTCCTCAAATAATAAAAATA TAAAAAGAACCCCCGGCGTAAAAAAAAAAAAAGAAGAGAGAAGGGGGAGCACACC CCCTCTTTTTTATTGTTGAAAAATATATAATAAAAA S-BPM- AAAAATTATAGGAATTCCGCATTTTCCATTTCTTCGAAATTTCTGCAACATCTAGAAC 001-HC- AATGGGCTGGTCCTGCATCATCCTGTTCCTTGTTGCTACTGCTACCGGCGTTCACTCTG KD For ATGTTCAACTTCTCGAGCAGGTTCAGCTTGTTCAGTCTGGTGTCGAGGTGAAGAAACC TGGCGCTTCTGTGAAAGTGAGCTGCAAGGCTTCTGGTTACACCTTCACCAACTACTAC ATGTACTGGGTGAGACAGGCTCCTGGTCAAGGTCTTGAATGGATGGGTGGTATCAAC CCTAGCAATGGTGGCACCAACTTCAACGAGAAGTTCAAGAACAGGGTGACCCTGACC ACCGATTCTTCTACTACTACCGCCTACATGGAACTGAAGTCCCTCCAGTTCGATGACA CCGCTGTTTACTATTGCGCTCGGAGGGATTACAGGTTCGATATGGGTTTCGATTACTG GGGTCAGGGTACCACCGTGACCGTTTCTTCTGCTAGCACCAAGGGACCTTCTGTGTTC CCTCTTGCTCCTTGCTCTAGGTCTACTTCTGAGTCTACTGCTGCTCTTGGTTGCCTGGT GAAGGATTACTTTCCTGAGCCTGTGACCGTGAGCTGGAATAGTGGTGCTCTTACTTCT GGCGTGCACACTTTCCCTGCTGTGCTTCAATCTAGCGGCCTGTACTCTCTTTCTTCTGT GGTGACTGTGCCGTCCAGCTCTCTTGGTACTAAGACCTACACTTGCAACGTGGACCAC AAGCCGTCTAATACCAAGGTGGACAAGCGGGTCGAGTCTAAGTATGGTCCTCCTTGT CCTCCATGTCCAGCTCCAGAATTTCTTGGTGGTCCTAGCGTGTTCTTGTTCCCGCCTAA GCCTAAGGATACCCTGATGATTTCTCGGACTCCTGAGGTTACCTGCGTTGTGGTTGAT GTGAGCCAAGAGGACCCAGAGGTTCAGTTCAACTGGTATGTGGATGGTGTTGAGGTG CACAACGCTAAGACTAAGCCTCGTGAGGAACAGTTCAACAGCACCTACAGGGTTGTG TCTGTGCTTACCGTGCTTCATCAAGATTGGCTGAACGGCAAAGAGTACAAGTGCAAG GTGAGCAACAAGGGCCTGCCGTCCTCTATGGAAAAAACATCTCTAAGGCTAAGGGCC AGCCTAGAGAACCGCAGTTTTCACTCTTCCCCCGAGCCAAAAAAGATGCCCCGAAAA CGGGGAACCTTACCTGCCTCTTGAGGGATTTTCCCCGCCCCTTTTTTTTGGTTGGGAAT TTTTTTGTTTCCCCCCAAAAAAAAAAAAAAAATCCCCCCCCCGGGGGAATATCAAGG TTGTTTTTTTTGTTGGAGGGCGTGCTGGTTTTTTTTATAGGGGGGGGGGGGGTTTTCCC CTTTTTGGGTGGGGGGCGGCCCCAACACCCAAATTTCTCCTCCCCCCCCACAAACAAC AGAAAGCGGTGCTGCCCCGCCTCCCCTTTTGGGAAGGCAT S-BPM- AACAAGCTATTATTTGGGCTTTATTACCTTCACTTTGTGATGTCACTTCGAGCTCTCAC 001-HC- AGCTCATCCTTCTCAGACTTTCCAAGGGACAGGGACAGAGACTTCTGGGTGTAGTGA KD Rev TTGTGCAGAGCCTCATGCATAACAGAGCAGCTGAACACGTTACCCTCCTGCCATCTTG ACTTATCCACGGTCAGCCTGCTGTACAGGAAGAATGAACCATCGGAATCCAGCACAG GAGGGGTAGTCTTGTAGTTGTTCTCGGGCTGACCATTAGACTCCCACTCGACAGCAAT GTCGCTAGGGTAAAATCCCTTCACGAGGCAGGTAAGGCTCACCTGATTCTTGGTCATC TCTTCTTGGCTCGGCGGAAGAGTGTAAACTTGCGGTTCTCTAGGCTGGCCCTTAGCCT TAGAGATGGTCTTTTCAATAGAGGACGGCAGGCCCTTGTTGCTCACCTTGCACTTGTA CTCTTTGCCGTTCAGCCAATCCTGATGAAGCACGGTAAGCACAGACACAACCCTGTA GGTGCTGTTGAACTGTTCCTCACGAGGCTTAGTCTTAGCGTTGTGCACCTCAACACCA TCCACATACCAGTTGAACTGAACCTCTGGGTCCTCTTGGCTCACATCAACCACAACGCDocket No.062822-501001WO AGGTAACCTCAGGAGTCCGAGAAATCATCAGGGTATCCTTAGGCTTAGGCGGGAACA AGAACACGCTAGGACCACCAAGAAATTCTGGAGCTGGACATGGAGGACAAGGAGGA CCATACTTAGACTCGACCCGCTTGTCCACCTTGGTATTAGACGGCTTGTGGTCCACGT TGCAAGTGTAGGTCTTAGTACCAAGAGAGCTGGACGGCACAGTCACCACAGAAGAA AGAGAGTACAGGCCGCTAGATTGAAGCACAGCAGGGAAAGTGTGCACGCCAGAAGT AAGAGCACCACTATTCCAGCTCACGGTCACAGGCTCAGGAAAGTAATCCTTCACCAG GCAACCAAGAGCAGCAGTAGACTCAGAAGTAGACCTAGAGCAAGGAGCAAGAGGGA ACACAGAAGGTCCCTTGGTGCTAGCAGAAGAAACGGTCACGGTGGTACCCTGACCCC AGTAATCGAAACCCATATCGAACCTGTAATCCCTCCGAGCGCATAGTAAACAGCGGT GTCATCCAACTGGAGGGACTTCATTTCATGTAGGCGGAATAATAAAAAAATCGGTGG TCAGGGTCCCCTGGTCTTGAACTTTCCCTTAAATTGGGGCCCCCTTGTTGGGGTTTAA CCCCCCCCCTTTAAAACTTTCCCAAAAGCCCTTTTCCCCCCACAAAATAATAAATTTG GGAAGGAAACCAAAAACTTTTCCCCCTTTTTAAAAACCCCGGTTTTTCTCCCCCCAAA AAAAAAATAACTCTCAAAAATAAAATAAAAAGACCCCGCGTCTTAAAAAAAAAAAG AGAGGGGCCCCCCCTTTTTATTTAATTTATAAAAAAGTTTTTTTTTGGT MGWSCIILFLVATATGVHSDVQLLEEIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSY SP-BPM- LHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRD 001-LC LPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C SP-BPM- MGWSCIILFLVATATGVHSDVQLLEQVQLVQSGVEVKKPGASVKVSCKASGYTFTNYY 001-HC MYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDT AVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS NTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDP EVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SP-BPM- MGWSCIILFLVATATGVHSDVQLLEQVQLVQSGVEVKKPGASVKVSCKASGYTFTNYY 001-HC- MYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDT KD AVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS NTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDP EVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKS EKDEL QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGG INPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDY WGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKT Pembroliz YTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDT umab HC LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYL ASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKV Pembroliz EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN umab LC SQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC Signal MGWSCIILFLVATATGVHS peptide Accession

Claims

Docket No.062822-501001WO No. P01750 Part of DVQLLE restriction enzyme sequence Pembroliz QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMG umab HC GINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYR Variable FDMGFDYWGQGTTVTVSS region Pembroliz EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLA umab LC SYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK Variable region Pembroliz umab HC NYYMY CDR1 Pembroliz umab HC GINPSNGGTNFNEKFKN CDR2 Pembroliz umab HC RDYRFDMGFDY CDR3 Pembroliz umab LC RASKGVSTSGYSYLH CDR1 Pembroliz umab LC LASYLES CDR2 Pembroliz umab LC QHSRDLPLT CDR3 MGWSCIILFLVATATGVHSDVQLLEQVQLVQSGVEVKKPGASVKVSCKASGY TFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTT SP- Keytruda- TAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSV HC(IgG4) FPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL with YSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLG Signal GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA peptide KTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG N297A QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKDocket No.062822-501001WO WHAT IS CLAIMED IS:

1. A recombinant antibody specific to PD-1, comprising one or more GlcNAc2Man3GlcNAc2 (GnGn) glycosylations.

2. A recombinant antibody specific to PD-1, comprising one or more antibody polypeptide sequences and a glycosylation pattern according to FIG.1B.

3. A recombinant antibody specific to PD-1, produced by expressing one or more antibody polypeptide sequences in a plant, wherein the antibody comprises one or more GlcNAc2Man3GlcNAc2 (GnGn) glycosylations.

4. The recombinant antibody of any one of claims 1 to 3, wherein the glycosylation is a N- glycosylation.

5. The recombinant antibody of any one of claims 1 to 4, wherein the glycosylation is at each Asn 297 in the Fc region of the antibody.

6. The recombinant antibody of any one of claims 1 to 5, wherein said glycosylation is essentially free of fucose.

7. The recombinant antibody of any one of claims 1 to 6, wherein the antibody comprises an Fc region selected from the group consisting of an IgGl, IgG2, IgG3 and IgG4 region.

8. The recombinant antibody of any one of claims 1 to 7, wherein the antibody polypeptide sequences lack an endoplasmic reticulum retention signal.

9. The recombinant antibody of any one of claims 1 to 7, wherein the antibody polypeptide sequences comprises an endoplasmic reticulum retention signal and the endoplasmic reticulum retention signal is SEKDEL.

10. The recombinant antibody of any one of claims 1 to 9, wherein the antibody is a monoclonal antibody.

11. The recombinant antibody of any one of claims 1 to 10, wherein the antibody is pembrolizumab.

12. The recombinant antibody of any one of claims 1 to 11, wherein the pembrolizumab comprises complementarity determining regions, heavy chain variable domain (VH), light chain variable domain (VL), or combinations thereof as described in Table 1.

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