WO2019055471A1

WO2019055471A1 - Methods for producing full-length antibodies

Info

Publication number: WO2019055471A1
Application number: PCT/US2018/050592
Authority: WO
Inventors: Andrew HORWITZ; Hailley Shannon WARBINGTON
Original assignee: Amyris, Inc.
Priority date: 2017-09-18
Filing date: 2018-09-12
Publication date: 2019-03-21

Abstract

The invention provides methods for producing full-length antibodies from genetically engineered non-pathogenic Trypanosomatidae host cells (e.g., Leishmania tarentolae host cells). The invention also provides methods for producing the genetically engineered non-pathogenic Trypanosomatidae host cells.

Description

METHODS FOR PRODUCING FULL-LENGTH ANTIBODIES FIELD OF THE INVENTION

[0001] The invention related to the production of high quality full-length antibodies by non- pathogenic Trypanosomatidae host cells (e.g., Leishmania tarentolae cells). BACKGROUND

[0002] Almost half of the top 10 best-selling drugs in 2016 were full-length monoclonal antibodies. Currently, the most commonly used method to produce monoclonal antibodies for therapeutic purposes is to use Chinese Hamster Ovary (CHO) cells or other mammalian cell lines, which are accepted by the industry and the Food and Drug Administration (FDA). In addition, a major advantage of CHO cells is that the glycosylation pattern on the secreted antibodies produced from CHO cells is the same as the mammalian glycosylation pattern, and thus, the antibodies are safe for use in patients. After decades of improvement, CHO cell line productivity is unlikely to be further improved significantly. Further, the demand for monoclonal antibodies is expected to surge as therapies for more widespread and chronic conditions (e.g. Alzheimer’s disease) are introduced. Some disadvantages of using CHO cells to produce antibodies include, e.g., a long timeline for production of a new antibody, and insufficient productivity and capacity to supply future needs. There is a need in the art for novel and efficient antibody production hosts that are able to produce safe antibodies for therapeutic purposes. SUMMARY

[0003] The invention provides methods for producing full-length antibodies from a genetically engineered non-pathogenic Trypanosomatidae host cell (e.g., a genetically engineered non-pathogenic Leishmania tarentolae host cell). The invention also provides methods for producing the genetically engineered non-pathogenic Trypanosomatidae host cell. [0004] In one aspect, the invention features a method for producing a genetically engineered non-pathogenic Trypanosomatidae host cell. The method includes: (a) contacting a non- pathogenic Trypanosomatidae host cell with a first heterologous nucleic acid sequence encoding an antibody light chain and a second heterologous nucleic acid sequence encoding an antibody heavy chain to produce the genetically engineered non-pathogenic Trypanosomatidae host cell; and (b) recovering the genetically engineered non-pathogenic Trypanosomatidae host cell, wherein the recovered, genetically engineered non-pathogenic Trypanosomatidae host cell includes the first and second heterologous nucleic acid sequences in its genome, and is capable of expressing the antibody light chain and the antibody heavy chain and secreting a full-length antibody therefrom. [0005] In some embodiments, the first and second heterologous nucleic acid sequences are joined through a linker sequence. In particular embodiments, the linker sequence is a cleavable linker sequence. In some embodiments, the first and second heterologous nucleic acid sequences each includes a secretion signal sequence. In particular embodiments, the first and second heterologous nucleic acid sequences are codon optimized according to codon frequencies of the host cell. In further embodiments, the first and second heterologous nucleic acid sequences are in an extrachromosomal expression vector or in an integration vector. [0006] In some embodiments, the host cell does not comprise a nucleic acid that encodes the full-length antibody Herceptin (trastuzumab) or the full-length antibody BIIB. In some embodiments, the host cell does not express the full-length antibody Herceptin (trastuzumab) or the full-length antibody BIIB. [0007] In some embodiments, the nucleic acid of interest excludes the nucleic acid that encodes the full-length antibody Herceptin (trastuzumab) or the full-length antibody BIIB. [0008] In some embodiments of this aspect, the host cell is Leishmania tarentolae. In particular embodiments, at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the population of antibodies or antibody fragments present in a culture medium produced from culturing the genetically engineered non-pathogenic Trypanosomatidae host cell are full-length antibodies. In some embodiments, the full-length antibody is BIIB or trastuzumab. In some embodiments, the genetically engineered non-pathogenic Trypanosomatidae host cell is capable of producing at least or about 4 mg/L of the full-length antibody. [0009] In other embodiments of this aspect, a low voltage electroporation is applied during contacting the non-pathogenic Trypanosomatidae host cell with the first heterologous nucleic acid sequence and the second heterologous nucleic acid sequence in step (a). [0010] In some embodiments of this aspect, the full-length antibody secreted by the genetically engineered, non-pathogenic Trypanosomatidae host cell is glycosylated. In particular embodiments, the full-length antibody is mannosylated. [0011] In another aspect, the invention features a method of producing a full-length antibody by: (a) providing a non-pathogenic Trypanosomatidae host cell including a first heterologous nucleic acid sequence encoding an antibody light chain and a second heterologous nucleic acid sequence encoding an antibody heavy chain; (b) culturing the host cell in a cell culture medium including a carbon source and a hemeprotein, wherein the cell culture medium is free of animal products; (c) expressing the antibody light chain and the antibody heavy chain under conditions that allow for the formation of the full-length antibody; and (d) recovering the full- length antibody, wherein the method produces a substantially homogenous population of antibodies. In some embodiments, the cell culture medium that is free of animal products contains peroxidase (e.g., peroxidase purified from horseradish). In some embodiments, the cell culture medium that is free of animal products contains catalase (e.g., catalase purified from Aspergillus niger). [0012] In some embodiments of this aspect, the non-pathogenic Trypanosomatidae host cell is Leishmania tarentolae. [0013] In particular embodiments of this aspect, the method produces a cell culture including at least or about 4 mg/L of the full-length antibody. In some embodiments, the full-length antibody is BIIB or trastuzumab. [0014] In some embodiments of this aspect, the method produces a cell culture which is capable of producing the full-length antibody with a single glycan species, e.g., Man3. [0015] In some embodiments of this aspect, the cell culture medium used in the method includes yeast extracts. In particular embodiments, the cell culture medium includes a catalase containing at least one heme group as the hemeprotein. [0016] In yet another aspect, the invention features a genetically engineered non-pathogenic Trypanosomatidae host cell. The genetically engineered non-pathogenic Trypanosomatidae host cell includes a first heterologous nucleic acid sequence encoding an antibody light chain and a second heterologous nucleic acid sequence encoding an antibody heavy chain in the cell’s genome. In some embodiments, the genetically engineered non-pathogenic Trypanosomatidae host cell is a genetically engineered non-pathogenic Leishmania tarentolae host cell. [0017] In a further aspect, the invention also features a cell culture medium that is free of any animal products. The cell culture medium may include a carbon source (e.g., yeast extracts) and a hemeprotein. The cell culture may further include one or more sugars. Examples of sugars that may be included in the cell culture medium include, but are not limited to, glucose, galactose, lactose, fructose, maltose, sucrose, cellulose, trehalose, starch, and amylose. In some embodiments, the cell culture medium that is free of animal products contains peroxidase (e.g., peroxidase purified from horseradish) as a source of heme. In some embodiments, the cell culture medium that is free of animal products contains catalase (e.g., catalase purified from Aspergillus niger) as a source of heme. [0018] In some embodiments of this aspect, the hemeprotein in the cell culture medium is a metalloprotein containing a heme prosthetic group. In a hemeprotein, the heme may remain bound to the heme carrier (e.g., a protein) covalently or non-covalently. The hemeprotein may serve diverse biological functions, such as oxygen and/or nitrogen transport, catalysis, and electron transport. A hemeprotein used in the cell culture medium may be derived from plants or other non-animal species, e.g., fungus, non-pathogenic bacteria, and non-pathogenic viruses. In some embodiments, the hemeprotein may be a leghemoglobin. In other embodiments, the hemeprotein may be a catalase containing at least one heme group derived from a fungus, e.g., Aspergillus niger. [0019] In some embodiments of this aspect, a cell culture medium that is free of any animal products may be used in methods of the invention for producing full-length antibodies from genetically engineered non-pathogenic Trypanosomatidae host cells (e.g., genetically engineered non-pathogenic Leishmania tarentolae host cells). The full-length antibodies produced therefrom may have identifiable glycan species, e.g., a single glycan species (e.g., Man3). BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG.1 is a schematic showing the structure of a full-length antibody. [0021] FIG. 2 is a Western blot showing similar banding patterns of a full-length BIIB antibody secreted from Leishmania tarentolae and a BIIB antibody secreted from CHO cells. [0022] FIG. 3 is a graph showing HPLC traces of glycan patterns of antibodies produced by Leishmania tarentolae. [0023] FIG. 4A is a Western blot showing similar banding patterns of a full-length trastuzumab antibody secreted from Leishmania tarentolae with those of standard full-length trastuzumab antibody. [0024] FIG.4B is a Western blot analysis of secreted BIIB and trastuzumab secreted from L. tarentolae. [0025] FIGS.5A and 5B illustrate L. tarentolae growth achieved on animal-free media. [0026] FIGS. 6A and 6B show MALDI-TOF data of the N-glycan profiles of BIIB and Herceptin produced from Leishmania. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] The methods described herein are directed to genetically engineering non-pathogenic Trypanosomatidae host cells by inserting both a heterologous nucleic acid sequence encoding an antibody light chain and another heterologous nucleic acid sequence encoding an antibody heavy chain into the host cell’s genome. The genetically engineered non-pathogenic Trypanosomatidae host cells (e.g., Leishmania tarentolae host cells) are able to produce full- length antibodies. [0028] Even though, CHO cells are the current choice host for production of monoclonal antibodies, the major limitations are that it takes a long timeline for production of a new antibody in CHO cells mainly due to long engineering cycle time (about 3 months) and its long doubling time (about 19-24 hours). Trypanosomatidae including Leishmania tarentolae is an emerging industrial host with potential for a wide variety of applications. Its doubling time is about 4.5 hours and its engineering cycle time is about 4 weeks. These features make Trypanosomatidae an attractive host for production of monoclonal antibodies due to its fast growth rate and less time it takes to engineer the strain. Furthermore, as illustrated in the present invention, Trypanosomatidae can provide economical expression and secretion of full- length antibodies with mammalian glycosylation patterns in suspension culture. I. Methods of Producing Genetically Engineered Non-Pathogenic Trypanosomatidae Host Cells

[0029] Much effort has gone into identifying and developing non-mammalian antibody production hosts. Production hosts such as filamentous fungi have been explored, but issues with poor antibody quality and aberrant glycosylation pattern have been difficult to overcome. To date, there have been no clinical trials involving antibodies produced from non-mammalian hosts. Trypanosomatidae is a family of flagellated protists under the class Kinetoplastidae. Protozoans in the Trypanosomatidae family are found in terrestrial and aquatic environments and characterized by the presence of an organelle called kinetoplast. Trypanosomatidae protozoans have been particularly valuable for the study of fundamental molecular and cellular phenomena, such as RNA editing, mRNA trans-splicing, glycosylphosphatidylinositol- anchoring of proteins, antigenic variations, and telomer organization. [0030] A unique feature of protozoans in the Trypanosomatidae family is that the protozoans have polycistronic transcription units. In these units, series of genes are arranged in tandem structures and transcribed by RNA polymerase as a single 50-100 kb transcript. Such transcript is then processed into single gene mRNAs by the addition of capped mini-exon at the 5' end followed by polyadenylation and cleavage of the 3' end. Further, gene regulation in Trypanosomatidae protozoans appears to happen almost exclusively at the post-translational level. In some embodiments, the oligosaccharide structures of glycoproteins produced from Trypanosomatidae protozoans are similar to those of mammalians. In few cases, e.g. Trypanosoma brucei, stage specific promoters such as VSG and PARP promoters are identified. These promoters recruit RNA polymerase I and not RNA polymerase II for protein encoding genes. It was shown that Trypanosomatidae protozoans are able to replicate both foreign and genome derived plasmids. The availability of genetic engineering methods in combination with advanced post-translational modifications makes members of the Trypanosomatidae family potential alternatives for biotechnological applications. Species of Trypanosomatidae that may be used as non-pathogenic host cells to produce full-length antibodies as described in the methods described herein include, but are not limited to, Blastocrithidia spp., Crithidia spp. (e.g., Crithidia fasciculate), Endotrypanum spp., Herpetomonas spp., Leishmania spp., Leptomonas spp. (e.g., Leptomonas collos, Leptomonas sp. Cfm, Leptomonas sp. Nfm, and Leptomonas seymouri), Paleoleishmania spp., Paleotrypanosoma spp., Phytomonas spp., Trypanosoma spp., and Wallaceina spp. (e.g., Wallaceina inconstans). In particular, the methods described herein may utilize Leishmania tarentolae as hose cells for full-length antibody production. [0031] Leishmania tarentolae is a eukaryotic flagellated unicellular parasite with a broad range of applications. It allows complex eukaryotic protein expression and secretion at high levels compared to other eukaryotes, and has the ability to post-translationally modify proteins with a glycosylation pattern similar to those of mammalian systems. Furthermore, Leishmania tarentolae can be cultured under fairly standard bioreactor conditions for protein production. All of these characteristics make Leishmania tarentolae a promising production system for full-length antibodies. As demonstrated in Examples 1-4, Leishmania tarentolae host cells were used to produce full-length antibody BIIB and full-length antibody trastuzumab with minimal degradation products or antibody fragments (i.e., which are not full-length antibody, heavy chain, or light chain). [0032] A non-pathogenic Trypanosomatidae host cell (e.g., a Leishmania tarentolae host cell) may be genetically engineered to produce full-length antibodies using methods described herein. The method may include: (a) contacting a non-pathogenic Trypanosomatidae host cell with a first heterologous nucleic acid sequence encoding an antibody light chain and a second heterologous nucleic acid sequence encoding an antibody heavy chain to produce a genetically engineered non-pathogenic Trypanosomatidae host cell; and (b) recovering the genetically engineered non-pathogenic Trypanosomatidae host cell. The genetically engineered non- pathogenic Trypanosomatidae host cell (e.g., the genetically engineered non-pathogenic Leishmania tarentolae host cell) includes the first and second heterologous nucleic acid sequences in its genome. Further, the genetically engineered non-pathogenic Trypanosomatidae host cell is capable of expressing the antibody light chain and the antibody heavy chain and secreting a full-length antibody therefrom. Moreover, the genetically engineered non-pathogenic Trypanosomatidae host cell is capable of secreting a population of antibodies or antibody fragments, wherein at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% the population of antibodies or antibody fragments that bind to goat anti-human IgG are full- length antibodies. In some embodiments, these percentages may be determined by weight when comparing the weight of the full-length antibodies to the weight of the population of antibodies or antibody fragments. In some embodiments, these percentages may be determined by densitometry of the Western blot bands. [0033] In some embodiments, a host cell line used for antibody production may be engineered to remove one or more proteases present in the cells. Removing one or more proteases may reduce protein degradation and improve protein stability of the proteins produced by the host cell. In some embodiments, the non-pathogenic Trypanosomatidae host cells (e.g., the non-pathogenic Leishmania tarentolae host cells) may be engineered to remove one or more proteases present in the host cells. Examples of proteases that may be removed from the host cell line include, but are not limited to, an aspartic protease, a cysteine protease, a threonine protease, a glutamic protease, a metalloprotease, an asparagine peptide lyase. However, in some embodiments, the deletion of one or more proteases from the host cell line may reduce the production efficiency and/or growth fitness of the cells. One of skill in the art has the knowledge and capabilities to determine if it is necessary to remove one or more proteases from the host cell line by molecular engineering techniques in order to improve the quality and stability of the secreted full-length antibodies. II. Methods For Producing Full-Length Antibodies

Expression vectors and construct design

[0034] In the present invention, a non-pathogenic Trypanosomatidae host cell (e.g., a Leishmania tarentolae host cell) is a vehicle that includes the necessary cellular components needed to express the full-length antibody from its corresponding nucleic acid sequence(s). One or more extrachromosomal expression vectors may be used to deliver the nucleic acid sequence(s) into the host cell. In some embodiments, a first heterologous nucleic acid sequence encoding an antibody light chain and a second heterologous nucleic acid sequence encoding an antibody heavy chain may each be inserted into an extrachromosomal expression vector. In other embodiments, the first and second heterologous nucleic acid sequences encoding the antibody light chain and the antibody heavy chain, respectively, may be inserted into the same extrachromosomal expression vector. In the embodiments where one extrachromosomal expression vector is used to express both the light chain and the heavy chain, additional 3’ and 5’ untranslated regions may be incorporated into the vector to ensure mRNA maturation and stability. [0035] In some embodiments, the nucleic acid sequence(s) encoding the light and heavy chains of the full-length antibody may be codon optimized according to codon frequencies of the host cell. Using the codon with the highest occurrence frequency in the host cell may reduce unwanted mutations and improve translation efficiency. The extrachromosomal expression vectors may also include appropriate expression control elements known in the art, including promoters, enhancers, selection markers, transcription terminators, and selectable markers. Methods for expressing therapeutic proteins are known in the art. See, for example, Paulina Balbas, Argelia Lorence (eds.) Recombinant Gene Expression: Reviews and Protocols (Methods in Molecular Biology), Humana Press; 2nd ed. 2004 edition (July 20, 2004); Vladimir Voynov and Justin A. Caravella (eds.) Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology) Humana Press; 2nd ed.2012 edition (June 28, 2012). The one or more extrachromosomal expression vectors containing the nucleic acid sequence(s) encoding the full-length antibody may can be introduced into the host cell by conventional techniques known in the art (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, etc.). [0036] An example of an extrachromosomal expression vector that may be used to integrate the nucleic acid sequence(s) encoding the full-length antibody into the chromosome of the non- pathogenic Trypanosomatidae host cell (e.g., a Leishmania tarentolae host cell) is LEXSY_I-3 expression vector (Jena Bioscience). Other extrachromosomal expression vectors that may be used in methods of the invention include, but are not limited to, pLEXSY, pLTEX, pJBS, pDEST-HisMBP, pDEST-periHisMBP, pGTvL1-SGC, pNH-TrxT, pBAD, pET 15, pTD, pCri, pBEST, pCS-PesaRlux, pCW-LIC, pPro, pdCas9, pDawn, pDusk, pJT106b3, pCph8, and pJT118. Linker

[0037] When one extrachromosomal expression vector contains both a light chain sequence and a heavy chain sequence, a linker sequence (e.g., a cleavable inker sequence) may be placed between the light chain sequence and the heavy chain sequence (e.g., SEQ ID NO: 1). For example, a nucleic acid sequence encoding the full-length antibody may include, in tandem series in this order from the 5’ end to the 3’ end, the light chain sequence, the linker sequence (e.g., a cleavable linker sequence), and the heavy chain sequence. In another example, the nucleic acid sequence encoding the full-length antibody may include, in tandem series in this order from the 5’ end to the 3’ end, the heavy chain sequence, the linker sequence (e.g., the cleavable linker sequence), and the light chain sequence. When the light chain sequence and the heavy chain sequence are each inserted in a separate extrachromosomal expression vector, a linker sequence may be placed between the secretion signal sequence in the light or heavy chain sequence. For example, a nucleic acid sequence encoding the light chain may include, in tandem series in this order from the 5’ end to the 3’ end, the secretion signal sequence, the linker sequence, and the light chain sequence. [0038] A linker may be a peptide having 3-200 (e.g., 3-150, 3-100, 3-80, 3-60, 3-40, 3-20, 3- 10, or 3-5) amino acids. Suitable peptide linkers are known in the art, and include, for example, peptide linkers containing flexible amino acid residues such as glycine and serine. A linker may be inserted between two polypeptides to provide space and/or flexibility between the two polypeptides. In certain embodiments, a peptide linker can contain motifs, e.g., multiple or repeating motifs, of GS, GGS, GGGGS (SEQ ID NO: 7), GGSG (SEQ ID NO: 8), or SGGG (SEQ ID NO: 9). In certain embodiments, a peptide linker can contain 2 to 12 amino acids including motifs of GS, e.g., GS, GSGS (SEQ ID NO: 10), GSGSGS (SEQ ID NO: 11), GSGSGSGS (SEQ ID NO: 12), GSGSGSGSGS (SEQ ID NO: 13), or GSGSGSGSGSGS (SEQ ID NO: 14). In certain other embodiments, a peptide linker can contain 3 to 12 amino acids including motifs of GGS, e.g., GGS, GGSGGS (SEQ ID NO: 15), GGSGGSGGS (SEQ ID NO: 16), and GGSGGSGGSGGS (SEQ ID NO: 17). In yet other embodiments, a peptide linker can contain 4 to 12 amino acids including motifs of GGSG (SEQ ID NO: 8), e.g., GGSG (SEQ ID NO: 8), GGSGGGSG (SEQ ID NO: 18), or GGSGGGSGGGSG (SEQ ID NO: 19). In other embodiments, a peptide linker can contain motifs of GGGGS (SEQ ID NO: 7), e.g., GGGGSGGGGSGGGGS (SEQ ID NO: 20). In other embodiments, a peptide linker can also contain amino acids other than glycine and serine, e.g., GENLYFQSGG (SEQ ID NO: 21), SACYCELS (SEQ ID NO: 22), RSIAT (SEQ ID NO: 23), RPACKIPNDLKQKVMNH (SEQ ID NO: 24), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 25), AAANSSIDLISVPVDSR (SEQ ID NO: 26), or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 27). [0039] In particular embodiments, a linker may be a cleavable linker that contains one or more elements that can be selectively cleaved, e.g., after a protein is formed. In some embodiments, a cleavable linker may be a self-cleavable linker, which may function by making the ribosome skip the synthesis of a peptide bond between a glycine amino acid and a proline amino acid near the C-terminus of the self-cleavable linker. Examples of self-cleavable linkers include, but are not limited to, RRKRGSGGGGEGRGSLLTCGDVEENPGPR (also referred to as 2A linker in Examples 1-4; SEQ ID NO: 4), GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 28), GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 29), GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 30), GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 31), EGRGSLLTCGDVEENPGP (SEQ ID NO: 32), ATNFSLLKQAGDVEENPGP (SEQ ID NO: 33), QCTNYALLKLAGDVESNPGP (SEQ ID NO: 34), and VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 35). [0040] In other embodiments, a linker may be a protease cleavable linker, which contains a protease cleavage site that is specifically recognized by a protease, e.g., a serine protease (e.g., factor Xa, enteropeptidase, proteinase K, chymotrypsin, trypsin, elastase, plasmin, thrombin, acrosomal protease, complement C1, keratinase, collagenase, fibrinolysin, and cocoonase), a cysteine protease (e.g., HRV3C protease, papain, bromelain, cathepsin, calpain, caspase-1, sortase, TEV protease, and hepatitis C virus peptidase 2), a threonine protease, an aspartic protease, a glutamic protease, a metalloprotease, and an asparagine peptide lyase. Examples of protease cleavable linkers include, but are not limited to, ENLYFQG (SEQ ID NO: 36; cleaved by TEV protease), LEVLFQGP (SEQ ID NO: 37, cleaved by HRV3C protease), IXGR (SEQ ID NO: 38, in which X is D or E; cleaved by factor Xa), DDDDKX (SEQ ID NO: 39, in which X is not P; cleaved by enterokinase). Secretion signal sequence

[0041] Moreover, one or more secretion signal sequences (e.g., two, three, four, five, six, seven, eight, nine, or ten secretion signal sequences) may be inserted in the extrachromosomal expression vector. The secretion signal sequence encodes a secretion signal peptide that is recognized by the molecular machinery of the host cell, which then secretes the protein from the cell. The choice of a secretion signal peptide may depend on the type of the host cell for antibody production. In some embodiments, the secretion signal peptide MASRLVRVLAAAMLVAAAVSVDAGA (SEQ ID NO: 3) may be used for full-length antibody production from a non-pathogenic Trypanosomatidae host cell (e.g., a Leishmania tarentolae host cell). Examples of other secretion signal peptides that may be used in methods described herein include, but are not limited to, MDWTWRILFLVAAATGTHA (SEQ ID NO: 40), MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO: 41), MASRLVRVLAAAMLVAAAVSVDA (SEQ ID NO: 42), and MASRLVRVLAAAMLVAAAVSVDAGASLD (SEQ ID NO: 43). In some embodiments, a secretion signal sequence may be placed at the 5’ end of the light chain sequence. In some embodiments, a secretion signal sequence may be placed at the 5’ end of the heavy chain sequence. In one example, when one extrachromosomal expression vector contains both a light chain sequence and a heavy chain sequence, e.g., SEQ ID NO: 1, a secretion signal sequence may be placed at the 5’ end of each of the light and heavy chain sequences. In some embodiments, having two secretion signal sequences in the extrachromosomal expression vector may promote antibody secretion. [0042] The sequence of SEQ ID NO: 1 below is an example of a nucleic acid sequence encoding both the BIIB antibody light chain and the BIIB antibody heavy chain, as well as a self-cleavable linker sequence and two secretion signal sequences. The sequence of SEQ ID NO: 1 may be inserted into an extrachromosomal expression vector, which can be transfected into the host cell. [0043] SEQ ID NO: 1 (bold: secretion signal sequence; not bold or underlined: BIIB antibody light chain sequence; squiggle underline: self-cleavable linker sequence; dash underline: BIIB antibody heavy chain sequence; 3’ end TAA: stop codon):

ATGGCTTCTCGTCTGGTGAGAGTCTTAGCCGCTGCCATGCTCGTGGCCGCAG CGGTTTCTGTTGACGCAGGCGCCGACATTCAGATGACCCAATCTCCATCGAGCC TTTCTGCTAGCGTGGGGGATCGCGTCACAATCACGTGTCGTGCATCCCAAGATATT CGTTACTATCTGAATTGGTATCAACAGAAGCCCGGTAAAGCGCCCAAACTGCTTAT CTACGTGGCCTCCAGCTTGCAATCTGGCGTTCCATCGCGTTTCAGCGGTAGCGGTT CGGGTACGGAGTTTACCCTCACGGTGTCGTCCCTGCAGCCAGAAGATTTCGCAACC TACTATTGCCTCCAGGTTTACTCCACTCCGCGTACTTTCGGCCAGGGGACGAAGGT CGAAATTAAACGCACAGTCGCCGCGCCTAGCGTCTTCATCTTCCCACCATCTGATG AGCAACTCAAGAGCGGCACGGCATCTGTTGTCTGCTTGCTGAATAATTTTTACCCA CGTGAGGCAAAGGTCCAGTGGAAAGTGGACAATGCTCTGCAGTCGGGCAACAGCC AGGAGTCGGTGACAGAGCAGGATTCGAAGGACTCGACCTACAGCCTGTCCAGCAC TCTGACTCTCTCTAAGGCAGACTATGAGAAACACAAGGTCTACGCTTGCGAGGTG ACGCATCAGGGGTTGTCGTCGCCAGTGACGAAGTCTTTCAACCGTGGTGAGTGCC GTCGCAAACGCGGCTCTGGTGGGGGTGGTGAAGGGCGTGGGTCTTTGCTGACGTG TGGCGATGTCGAGGAGAACCCAGGGCCACGTATGGCATCCCGCCTGGTGCGTG TTCTCGCAGCAGCTATGCTCGTGGCAGCGGCAGTTTCTGTTGACGCCGGCGC CGAGGTGCAGCTGGTTGAGTCTGGCGGTGGCCTGGCTAAACCCGGTGGCTCCTTG CGTCTGAGCTGTGCGGCTAGCGGGTTTCGGTTCACCTTCAACAACTACTACATGGA CTGGGTCCGTCAGGCTCCTGGTCAGGGTCTTGAATGGGTGTCCCGCATCTCGTCGA GCGGTGATCCGACTTGGTATGCTGACTCCGTGAAAGGCCGCTTCACCATTAGCCGC GAGAACGCCAAAAATACGCTGTTCCTGCAGATGAACTCGCTCCGGGCAGAAGACA CGGCTGTCTACTACTGCGCGAGCTTGACGACAGGGTCCGACTCCTGGGGCCAAGG TGTTTTGGTCACGGTTAGCTCGGCATCTACCAAGGGCCCTTCCGTCTTTCCGCTGGC CCCTTCGAGCAAATCTACAAGCGGGGGTACAGCCGCTCTTGGGTGCCTTGTGAAG GACTACTTCCCGGAGCCTGTGACAGTGTCGTGGAACAGCGGTGCGCTTACGTCGG GTGTGCACACCTTCCCCGCGGTCTTGCAATCCAGCGGTCTGTACTCCTTGAGCAGC GTGGTCACCGTCCCTTCCTCCTCGCTGGGCACCCAGACTTACATCTGTAACGTGAA CCATAAACCATCGAACACGAAAGTGGATAAGAAGGTGGAGCCCAAGAGCTGTGA TAAAACACACACTTGTCCCCCATGTCCGGCTCCAGAGTTGCTTGGCGGGCCGTCTG TGTTTCTCTTTCCCCCGAAGCCGAAGGATACACTGATGATCTCTCGCACGCCGGAG GTTACGTGCGTGGTCGTGGACGTGAGCCACGAGGATCCTGAGGTCAAATTTAACT GGTACGTGGACGGCGTCGAGGTCCACAACGCGAAGACGAAACCACGGGAAGAAC AGTATAATTCCACATACCGTGTGGTCAGCGTGCTCACCGTGCTCCACCAGGACTGG CTCAATGGTAAGGAGTACAAATGCAAGGTGTCCAACAAGGCCTTGCCCGCGCCGA TTGAAAAGACTATTTCGAAGGCAAAAGGCCAGCCTCGGGAACCTCAGGTGTACAC GCTGCCACCGTCGCGTGACGAGCTCACCAAGAACCAGGTGTCTCTCACGTGCCTCG TGAAGGGGTTCTACCCCAGCGACATCGCGGTCGAATGGGAGAGCAACGGTCAGCC GGAGAATAACTATAAGACCACGCCCCCAGTGCTCGATTCGGACGGCTCTTTTTTCC TCTATTCTAAGCTGACGGTCGATAAGAGCCGTTGGCAGCAGGGCAACGTGTTTTCT TGCTCGGTCATGCACGAGGCCCTGCACAACCACTATACACAGAAGTCGCTCTCCCT TTCTCCCGGGAAATAA. [0044] The sequence of SEQ ID NO: 2 below is the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 1. [0045] SEQ ID NO: 2 (bold: secretion signal; not bold or underlined: BIIB antibody light chain; squiggle underline: self-cleavable linker; dash underline: BIIB antibody heavy chain): MASRLVRVLAAAMLVAAAVSVDAGADIQMTQSPSSLSASVGDRVTITCRASQDIRY YLNWYQQKPGKAPKLLIYVASSLQSGVPSRFSGSGSGTEFTLTVSSLQPEDFATYYCL QVYSTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGECRRKRGSGGGGEGRGSLLTCGDVEENPGPRMASRLVRVLAAAMLVA AAVSVDAGAEVQLVESGGGLAKPGGSLRLSCAASGFRFTFNNYYMDWVRQAPGQGL EWVSRISSSGDPTWYADSVKGRFTISRENAKNTLFLQMNSLRAEDTAVYYCASLTTGS DSWGQGVLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. Cell culture medium

[0046] Non-pathogenic Trypanosomatidae host cells (e.g., Leishmania tarentolae host cells) transfected with one or more the extrachromosomal expression vectors and/or integration plasmids containing the nucleic acid sequence(s) encoding the full-length antibody (e.g., SEQ ID NO: 1), one or more secretion signal sequences, and a cleavable linker sequence, may be cultured in an appropriate cell culture medium that promotes full-length antibody expression, assembly, and secretion. In particular embodiments, the cell culture medium used in methods of the invention is free of animal products. Cell culture media containing animal products (e.g., mammalian proteases, serum proteins, growth promoters and/or inhibitors) may introduce unwanted issues, such as protein degradation, contamination, protein aggregation, and abnormal protein growth rate, into the antibody production process. Moreover, cell culture media containing animal products may further require downstream processing to remove serums for antibody isolation. Using a cell culture medium free of animal products may reduce or eliminate one or more of these issues, leading to an increased yield of antibodies that are more homogenous. [0047] A cell culture medium free of animal products used in the methods of the invention may include a carbon source and a hemeprotein. In some embodiments, a cell culture medium may include one or more sugars. A carbon source may be a yeast extract. Examples of sugars that may be included in the cell culture medium include, but are not limited to, glucose, galactose, lactose, fructose, maltose, sucrose, cellulose, trehalose, starch, and amylose. A hemeprotein is a metalloprotein containing a heme prosthetic group. The heme may remain bound to a heme carrier (e.g., a protein) covalently or non-covalently to form a hemeprotein. The hemeprotein may serve diverse biological functions, such as oxygen and/or nitrogen transport, catalysis, and electron transport. A hemeprotein used in the cell culture medium may be derived from plants or other non-animal species, e.g., fungus, non-pathogenic bacteria, and non-pathogenic viruses. In some embodiments, the hemeprotein may be a leghemoglobin. In other embodiments, the hemeprotein may be a catalase containing at least one heme group derived from a fungus, e.g., Aspergillus niger. In some embodiments, the cell culture medium that is free of animal products contains peroxidase (e.g., peroxidase purified from horseradish) as a source of heme. In some embodiments, the cell culture medium that is free of animal products contains catalase (e.g., catalase purified from Aspergillus niger) as a source of heme. A cell culture medium that is free of any animal products may be used to produce full-length antibodies from genetically engineered non-pathogenic Trypanosomatidae host cells (e.g., genetically engineered non-pathogenic Leishmania tarentolae host cells). The full-length antibodies produced therefrom may have a glycosylation pattern substantially equivalent to the glycosylation pattern of the same full-length antibodies produced from mammalian cells (e.g., CHO cells). [0048] The invention includes a cell culture for full-length antibody production that includes a non-pathogenic Trypanosomatidae host cell (e.g., a Leishmania tarentolae host cell) transfected with one or more expression vectors containing a first heterologous nucleic acid sequence encoding an antibody light chain and a second heterologous nucleic acid sequence encoding an antibody heavy chain, as well as one or more secretion signal sequences and one or more linker sequences. [0049] In certain embodiments, the cell culture also includes a cell culture medium that is free of animal products and includes a carbon source (e.g., a yeast extract) and a hemeprotein (e.g., a leghemoglogin or a catalase containing heme group(s) derived from a fungus, e.g., Aspergillus niger). In some embodiments, the cell culture medium that is free of animal products contains peroxidase (e.g., peroxidase purified from horseradish) as a source of heme. In some embodiments, the cell culture medium that is free of animal products contains catalase (e.g., catalase purified from Aspergillus niger) as a source of heme. The yeast extract provides, among others, a carbon source and a nitrogen source. In some embodiments, the cell culture medium also includes another carbon source, such as glucose. In particular embodiments, the cell culture medium excludes hemin derived from an animal source. In some embodiments of this cell culture, at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the population of secreted antibodies or antibody fragments (which bind to goat anti-human IgG) from Trypanosomatidae host cell (e.g., Leishmania tarentolae host cell) are full-length antibodies. In certain embodiments, the substantially homogenous population of antibodies may include, at least 40%, full-length antibodies. The substantially homogenous population of antibodies may include, less than 30%, degradation products (e.g., truncated antibodies, antibody fragments). In some embodiments, these percentages may be determined by weight when comparing the weight of the full-length antibodies to the weight of the population of antibodies or antibody fragments. In some embodiments, these percentages may be determined by densitometry of the Western blot bands. In some embodiments, the cell culture may contain at least 4 mg/L (e.g., at least 4.5 mg/L, at least 5 mg/L, at least 5.5 mg/L, at least 6 mg/L, at least 6.5 mg/L, at least 7 mg/L, at least 7.5 mg/L, at least 8 mg/L, at least 8.5 mg/L, at least 9 mg/L, at least 9.5 mg/L, or at least 10 mg/L) of the full-length antibody secreted by the host cell. In some embodiments, the cell culture may contain at least 4 mg/L but less than 50 mg/L of the full- length antibody secreted by the host cell, or any titers between these ranges. In some embodiments, the cell culture may contain at least 1 mg (e.g., at least 1.5 mg/L, at least 2 mg/L, at least 2.5 mg/L, at least 3 mg/L, at least 3.5 mg/L, at least 4 mg/L or more) of the full- length antibody secreted by the host cell. In some embodiments, the cell culture may contain at least 1 mg/L but less than 50 mg/L secreted by the host cell, or any titers within these ranges. In some embodiments, the cell culture may contain at least 1 mg/L but less than 100 mg/L, 1 g/L, 10 g/L, 50 g/L, or any titers within these ranges. [0050] In certain embodiments, the cell culture medium that is free of animal products described herein (e.g., a cell culture medium comprising a carbon source (e.g., yeast extracts) and a hemeprotein (e.g., a hemeprotein derived from plants or other non-animal species (e.g., fungus, non-pathogenic bacteria, and non-pathogenic viruses); a leghemoglobin; a catalase containing at least one heme group derived from a fungus, e.g., Aspergillus niger)) may be used to produce any suitable antibodies, i.e., full-length antibodies or antigen-binding fragments thereof. An antibody that may be produced using a cell culture medium free of animal products refers to a protein functionally defined as a binding protein and structurally defined as comprising an amino acid sequence that is recognized by one of skill as being derived from a variable region of an immunoglobulin encoding gene. The term encompasses intact polyclonal antibodies, intact monoclonal antibodies, single chain antibodies, multispecific antibodies such as bispecific antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, and human antibodies. The term “antibody,” as used herein, also includes antibody fragments that retain binding specificity, including but not limited to Fab, F(ab’)₂, Fv, scFv, and bivalent scFv. An antibody can consist of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as 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. Each of the heavy and light chains contains three complementarity determining regions (CDRs), which are the three hypervariable regions in each chain that interrupt the four framework regions established by the light and heavy chain variable regions. The CDRs are primarily responsible for antibody binding to an epitope of an antigen. 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) and“variable heavy chain” (VH) refer to these light and heavy chains, respectively. [0051] The cell culture medium that is free of animal products described herein (e.g., a cell culture medium comprising a carbon source (e.g., yeast extracts) and a hemeprotein (e.g., a hemeprotein derived from plants or other non-animal species (e.g., fungus, non-pathogenic bacteria, and non-pathogenic viruses); a leghemoglobin; a catalase containing at least one heme group derived from a fungus, e.g., Aspergillus niger)) may also be used to produce antigen-binding fragments. Antigen-binding fragments refer to an antibody fragment including, for example, a diabody, a Fab, a Fab’, a F(ab’)2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv’), a disulfide stabilized diabody (ds diabody), a single-chain Fv (scFv), an scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment binds. [0052] In certain embodiments, a cell culture medium free of animal products (e.g., a cell culture medium comprising a carbon source (e.g., yeast extracts) and a hemeprotein (e.g., a hemeprotein derived from plants or other non-animal species (e.g., fungus, non-pathogenic bacteria, and non-pathogenic viruses); a leghemoglobin; a catalase containing at least one heme group derived from a fungus, e.g., Aspergillus niger)) may be used to produce full-length antibodies or antigen-binding fragments thereof from genetically engineered non-pathogenic Trypanosomatidae host cells (e.g., genetically engineered non-pathogenic Leishmania tarentolae host cells). [0053] Furthermore, in certain embodiments, the invention features a method of producing an antibody or an antigen-binding fragment thereof by: (a) providing a non-pathogenic Trypanosomatidae host cell including a first heterologous nucleic acid sequence encoding an antibody light chain or a fragment thereof and a second heterologous nucleic acid sequence encoding an antibody heavy chain or a fragment thereof; (b) culturing the host cell in a cell culture medium including a carbon source and a hemeprotein, wherein the cell culture medium is free of animal products; (c) expressing the antibody light chain or the fragment thereof and the antibody heavy chain or the fragment thereof under conditions that allow for the formation of the antibody or the antigen-binding fragment thereof; and (d) recovering the antibody or the antigen-binding fragment thereof, wherein the method produces a substantially homogenous population of antibodies or a substantially homogenous population of antigen-binding fragments. In some embodiments, the cell culture medium that is free of animal products contains peroxidase (e.g., peroxidase purified from horseradish) as a source of heme. In some embodiments, the cell culture medium that is free of animal products contains catalase (e.g., catalase purified from Aspergillus niger) as a source of heme. Antibody isolation and purification

[0054] Full-length antibodies may be isolated and purified by any method known in the art of protein isolation and purification. In some embodiments, full-length antibodies secreted by the Trypanosomatidae host cell (e.g., Leishmania tarentolae host cell) in the cell culture may be isolated by centrifugation to remove the cells and filtration to isolate the full-length antibodies from the supernatant. Further, liquid chromatography and/or densitometry of Western blot bands may be used assess the quality of the full-length antibodies. In some embodiments, full-length antibodies may further be purified, for example, by chromatography (e.g., size-exclusion column chromatography, ion exchange, and affinity column chromatography (e.g., Protein A affinity column)), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. In Protein A affinity column chromatography, Protein A ligands interact with the Fc region of the full-length antibodies, making Protein A affinity column chromatography a highly selective capture process that is able to remove most of the host cell proteins. For example, in some embodiments, if necessary, full-length antibodies may be isolated and purified by appropriately selecting and combining affinity columns such as Protein A column with chromatography columns, filtration, ultra filtration, salting-out and dialysis procedures (see, e.g., Process Scale Purification of Antibodies, Uwe Gottschalk (ed.) John Wiley & Sons, Inc., 2009; and Subramanian (ed.) Antibodies-Volume I-Production and Purification, Kluwer Academic/Plenum Publishers, New York (2004)). In other embodiments, full-length antibodies may be conjugated to purification sequences, such as a peptide to facilitate purification. An example of a purification sequence is a hexa-histidine peptide, which binds to nickel-functionalized agarose affinity column with micromolar affinity. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin“HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein. III. Full-Length Antibodies

[0055] A full-length immunoglobulin (Ig) antibody has two identical heavy chain polypeptides, two identical light chain polypeptides, disulfide linkages connecting the two light chain polypeptides, and a glycosylation pattern (see, e.g., FIG. 1). As shown in FIG. 1, each heavy chain includes a constant region (e.g., CH1, CH2, and CH3) and a variable region (e.g., V_H) joined by a hinge region. The two constant regions of the two heavy chains form an Fc domain (e.g., Fc). Each light chain includes a constant region (e.g., CL) and a variable region (e.g., V_L). An antigen-binding fragment (Fab) is a region on an antibody that binds to antigens. It is composed of one constant domain and one variable domain of each of the heavy and light chain (e.g., V_H, C_H1, V_L, and C_L). A variable fragment (Fv) refers to a fragment containing a variable region of one heavy chain and a variable region of one light chain (e.g., VH and VL). Further, two constant regions of the two heavy chains form the Fc domain of the antibody (e.g., CH2 and CH3 from each of the two heavy chains). A full-length antibody may be of any isotype (e.g., IgA, IgD, IgE, IgG, and IgM), which is defined by the heavy chain of the antibody. [0056] The vast majority of approved antibody therapies are for full-length antibodies, which often have much longer serum half-life and engage the immune response more efficiently than antibody fragments. Antibody fragments tend to be rapidly cleared from the patient, form unwanted aggregates, and lack full or partial glycosylation pattern. Methods to extend the half-life of antibody fragments have been explored, such as conjugation to albumin or PEG units. However, these methods are likely to increase the manufacture complexity and cost of antibody production. Moreover, the lack of an Fc domain in antibody fragments may further increase the risk of protein aggregation during production. Without an Fc domain, the antibody fragments also are not able to induce Fc-mediated functions, such as antibody- dependent cell-meditated cytotoxicity or complement-dependent cytotoxicity. [0057] The methods of the invention describe the production of full-length antibodies from a genetically engineered non-pathogenic Trypanosomatidae host cell (e.g., a genetically engineered non-pathogenic Leishmania tarentolae host cell) using a cell culture medium that is free of any animal products. The host cell is able to produce a substantially homogenous population of antibodies having less than 30% degradation products and at least 40% full- length antibodies. As shown in FIGS. 2 and 4, Leishmania tarentolae secreted the full-length BIIB antibody and the full-length Herceptin (trastuzumab) antibody, which exhibited a banding pattern essentially indistinguishable from the corresponding standard BIIB antibody and Herceptin antibody secreted from CHO cells. Moreover, there are virtually no degradation products of the full-length antibody. Further, the full-length antibodies produced from methods described herein have a single glycan species, e.g., Man3, which is mammalian-like. This is an acceptable glycan for further engineering. As demonstrated in Examples 1-4, the production of high quality, full-length antibodies from Leishmania tarentolae establishes the protozoan as a superior replacement for mammalian cell line-based production systems (e.g., CHO cells). IV. Definitions

[0058] As used herein, the term“full-length antibody” refers to an antibody having a structure substantially similar to a native antibody structure. A full-length antibody includes four polypeptides– two light chains and two heavy chains joined by disulfide bonds to form a “Y” shaped molecule. Each heavy chain includes a constant region and a variable region join by a hinge region. The two constant regions of the two heavy chains form an Fc domain. A full-length antibody may be of any isotype (e.g., IgA, IgD, IgE, IgG, and IgM), which is defined by the heavy chain of the antibody. [0059] As used herein, the term“non-pathogenic” refers to an organism that does not cause any disease and/or is non-infectious to humans. [0060] As used herein, the term“host cell” refers to a vehicle that includes the necessary cellular components, e.g., organelles, needed to express proteins, e.g., full-length antibodies, from their corresponding nucleic acids. The nucleic acids are typically included in extrachromosomal expression vectors that can be introduced into the host cell by conventional techniques known in the art (transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, etc.). As described herein, a genetically engineered non- pathogenic Trypanosomatidae host cell (e.g., a genetically engineered non-pathogenic Leishmania tarentolae host cell) is used to express full-length antibodies. [0061] As used herein, the term“heterologous nucleic acid sequence” refers to a nucleic acid sequence that is not originally present in the endogenous chromosome of the host cell. A heterologous nucleic acid sequence may inserted into the endogenous chromosome of the host cell using molecular cloning techniques. [0062] As used herein, the term“non-degraded” antibodies refer to antibodies associated with the top three gel bands visible on the Western blot (see, e.g., FIGS.2 and 4), for example, a full-length antibody, a half of a full-length antibody (i.e, a single heavy chain and a single light chain), and a heavy chain. As used herein, the term“degraded” antibodies refer to the rest of gel bends (bands other than the top three gel bands) (see, e.g., FIGS. 2 and 4) on the Western blot associated with fragments smaller than non-degraded antibodies. [0063] As used herein, the term“substantially homogenous population of antibodies” refers to a population of antibodies containing full-length antibodies that form at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the entire population of antibodies (i.e., non-degraded antibodies and degraded antibodies or fragments that bind to anti-IgG, e.g., goat anti-human IgG). In some embodiments, less than 20% (e.g., less than 20%, less than 10%, or less than 5%) of the substantially homogenous population of antibodies contains degraded antibodies or antibody fragments. In some embodiments, at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the substantially homogenous population of antibodies are full-length antibodies that have the same three-dimensional structure. In some embodiments, these percentages may be determined by weight when comparing the weight of the full-length antibodies to the weight of the population of antibodies or antibody fragments. In some embodiments, these percentages may be determined by densitometry of the Western blot bands. In some embodiments, the full- length antibody represents at least 40%, 50%, 70%, 80%, 90%, or 95% of all visible antibody bands (the sum of all bands including full-length antibody) according to Western blot densitometry. In some embodiments, the full-length antibodies have identifiable glycan species, e.g., Man3. The Man3 is mammalian-like, but not see in the HPLC trace from the CHO cells (the CHO trace omitted in FIG.3). [0064] As used herein, the term“linker” refers to a linkage between two polypeptides, e.g., a light chain and a heavy chain. A linker may be a peptide linker having 3-200 amino acids occurring between two polypeptides to provide space and/or flexibility between the two polypeptides. A linker may also be a self-cleavable linker or a protease cleavable linker. A self-cleavable linker refers to a linker that can be cleaved without the need of a protease, i.e., by making the ribosome skip the synthesis of a peptide in the self-cleavable linker. A protease cleavable linker refers to a linker containing one or more elements, e.g., cleavage sites, that can be selectively cleaved by a protease. [0065] As used herein, the term“codon optimized” refers to a nucleic acid sequence in which all the codons in the sequence are matched to the codons with the highest occurrence or usage frequency within the degenerate codons in the host cell. [0066] As used herein, the term“free of animal products” refers to a culture medium which does not include any proteins present in a mammalian cell culture medium, such as a Brain Heart Infusion (BHI) medium. A cell culture medium free of animal products does not contain, e.g., mammalian proteases, mammalian serum proteins, and mammalian growth promoters and/or inhibitors. [0067] As used herein, the term“carbon source” refers to a nutrient in the cell culture medium that provides carbons in the culturing of the host cell. [0068] As used herein, the term“hemeprotein” refers to a protein containing a heme group that functions to, e.g., transport oxygen and/or nitrogen, catalyze various cellular reactions, and shuttle electrons among cellular proteins. A hemeprotein used in a cell culture medium free of animal products may be a leghemoglobin or may be derived from a fungus, e.g., a catalase containing at least one heme group from Aspergillus niger. In some embodiments, the cell culture medium that is free of animal products contains peroxidase (e.g., peroxidase purified from horseradish) as a source of heme. In some embodiments, the cell culture medium that is free of animal products contains catalase (e.g., catalase purified from Aspergillus niger) as a source of heme. [0069] As used herein, hemin is an iron containing porphyrin. More specifically, it is protoporphyrin IX containing a ferric iron ion (heme B) with a chloride ligand. The commercially available hemin is currently extracted from an animal blood. [0070] As used herein, the term“polynucleotide” refers to an oligonucleotide, or nucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single- or double-stranded, and represent the sense or anti-sense strand. A single polynucleotide is translated into a single polypeptide. [0071] As used herein, the term“polypeptide” describes a single polymer in which the monomers are amino acid residues which are joined together through amide bonds. A polypeptide is intended to encompass any amino acid sequence, either naturally occurring, recombinant, or synthetically produced. EXAMPLES

Example 1: Production of Full-Length Antibody in Leishmania tarentolae and its Secretion

[0072] This example demonstrates that a high quality, full-length antibody can be produced and secreted from Leishmania tarentolae without producing much degradation products. Parent Host Cell: LEXSY host T7-TR

[0073] The parent host cell LEXSY host T7-TR was obtained from Jena Biosicence (Cat. No. LT-110; Jena/Thüringen, Germany). In the LEXSY host T7-TR, T7 RNA polymerase and TET operator genes were constitutively integrated into the 18s rRNA locus (see description in Cat. No. LT-110, Jena Bioscience). Maintenance of the T7 and TET operator genes is facilitated by supplementation with Nourseothricin and Hygromycin at final concentrations of 100µg/ml each. T7 RNA polymerase and TET operator allow for expression of target genes by tetracycline induction. The average growth rate (hr^-1) for L. tarentolae is known to be about 0.074. LEXSY_I-3 Expression Vector

[0074] LEXSY_I-3 expression vector (Jena Bioscience) can be used to integrate a target gene into the ornithine decarboxylase (odc) locus of chromosome 12 of Leishmania tarentolae. T7 promotor drives expression of the target gene and antibiotic resistance gene, with three untranslated regions offering splicing signals for posttranslational mRNA modifications. Expression cassette is flanked by 5’ and 3’ odc regions for homologous recombination into the host chromosome. Generation of Antibody Construct

[0075] The amino acid sequence of the full-length antibody BIIB construct is shown as the sequence of SEQ ID NO: 2. The amino acid sequence of SEQ ID NO: 2 includes, in the order from the N-terminus to the C-terminus, a secretion signal sequence (SEQ ID NO: 3), a light chain sequence of BIIB antibody, 2A linker sequence (SEQ ID NO: 4), a secretion signal sequence (SEQ ID NO: 3), and a heavy chain sequence of BIIB antibody. The 2A linker sequence is a“self-cleaving” peptide. (Addgene plasmid 101: Multicistronic Vectors, Sept. 2014). SEQ ID NO: 5 and SEQ ID NO: 6 further show the nucleic acid sequence and amino acid sequence, respectively, of full-length antibody Herceptin (trastuzumab) used in Example 1. The sequences also include the secretion signal sequence and the 2A linker sequence used in the full-length antibody BIIB. [0076] Antibody constructs were codon optimized according to Leishmania tarentolae codon frequency tables (Codon Usage Database by Kazusa), and DNA sequences were cloned into the LEXSY_I-3 expression vector (Jena Bioscience) containing the S. hindustins gene conferring resistance to zeocin using standard procedures. Plasmids were linearized using restriction endonucleases and purified by gel electrophoresis. 0.4-5.0 µg of linear DNA was introduced into Leishmania tarentolae cells (approximately 10⁸ cells/mL density) by low voltage electroporation (450V, 450 µF, pulse time monitored at 5-6ms). Electroporated cells were immediately transferred to fresh BHI (brain-heart-infusion-based) media supplemented with hemin and maintenance antibiotics (Nat, Hyg, and Pen-Strep), and allowed to recover in ventilated tissue culture flasks overnight. Cells were then plated on solid BHI agar supplemented with hemin, maintenance antibiotics, and zeocin (100 µg/ml) for selective pressure. The BHI media was prepared from LEXSY BHI-powered media kit (Jena Bioscience). Strain Selection

[0077] Colonies were picked in 2.2 mL 96-well plates and suspended in 1.0 mL fresh BHI (brain heart infusion), YE (2.4% Bacto yeast extract, 1% glucose or 1% fructose, 90 mM phosphate buffer, pH 7.5), or YPD (2% glucose) containing 5 μg/mL hemin, 100 μg/mL zeocin, 100 μg/mL Hygromycin B, 100 μg/mL nourseothricin, and 0.5% v/v 200x Pen-Strep stock solution. Cultures were allowed to grow for 72 hours at 26°C with occasional agitation before being spun down (2,000 g for 5 min) and resuspended in 1.0 mL of the same media as above with 100 µg/mL tetracycline as inducer and grown at 26°C for an additional 48 hours. Cultures were centrifuged at 2,000 g for 5 min and 500 µL of clarified supernatants were filtered through a nitrocellulose membrane for future analyses. Top antibody producers were run in 250 mL shake flasks containing 50 mL media and shaken at 100 rpm to achieve large volumes for N-glycan analysis. For example, cultures were sedimented and 500 µL of clean supernatant filtered through a nitrocellulose membrane for dot blot analysis (Goat anti-human IgG). Highest fluorescing clones were selected for further downstream protein analysis. Protein A purification

[0078] Supernatant samples from antibody production cultures were purified and concentrated using Protein A tip columns (PhyNexus, San Jose, CA, USA). Semi-quantitative antibody titer measurement by Dot Blot

[0079] Dot blot analysis was carried out using the Minifold I 96-Well System (GE Healthcare, Little Chalfont, UK) according to the manufacturer protocol. Supernatants were collected from cultures grown under production conditions. Detection was performed using IRDye® 800CW goat anti-human IgG (H + L) antibody (LI-COR, Lincoln, NE, USA) as both the primary and secondary antibody and imaged on the Odyssey Infrared Imaging System (LI- COR, Lincoln, NE, USA). EndoH treatment

[0080] Endoglycosidases treatment was done using Endo Hf (New England Biolabs, Cat. No. P0703S) according to the manufacturer’s instructions. For non-reducing samples, 1X Glycoprotein Denaturing Buffer was replaced with 5% SDS solution. Strain Cultivation and Sample Preparation for Western Blot

[0081] Strains were grown in pre-culture media (BHI, hemin, zeocin, and maintenance antibiotics) for 72 hours as static suspension cultures. Cells were then sedimented, resuspended in production media (pre-culture media + 100 µg/mL tetracycline) and incubated with continuous agitation. After a 48-hour production period, cells were sedimented and supernatant was run through a 0.2 µm filter. 7.5 µL of clean supernatant was boiled with 2.5 µL of Western blot loading buffer and loaded directly onto tris-acetate protein gels by standard procedures. The antibodies were probed with goat anti-human IgG. [0082] More specifically, all monoclonal antibody samples were mixed with NuPAGE LDS Sample Buffer (Thermofisher, Cat. No. NP008) and denatured at 70°C for 10 min before running non-reduced samples on 3-8% Tris-Acetate precast protein gels (Thermofisher, Cat. No. EA0375). For reduced samples, NuPAGE Sample reducing Buffer (Thermofisher, Cat. No. NP009) was used as reducing agent. Reduced samples were denatured at 70°C for 10 minutes and then run on 4-12% Bis-Tris precast protein gels (Thermofisher, Cat. No. NP0321). For investigation BIIB degradation by intracellular cell lysate, samples were run on a 48-well E-PAGE gel using the iBlot system (Thermofisher). For Western Blot analysis, Goat anti- human IgG (H + L) (LiCor, Cat. No. 925-32232) was used at a 1:10,000 dilution to detect heavy chain, light chain or full length antibody. Western Blot Results– BIIB antibodies

[0083] The results of the Western blot for antibodies BIIB are shown in FIG.2. As shown in FIG. 2, Leishmania tarentolae secreted the full-length BIIB antibody, which exhibited a banding pattern indistinguishable from the BIIB antibody secreted from CHO cells. The top band represents the full-length BIIB antibody; other bands represent fragments of the full- length antibody. For example, the second band from the top may be a half of the full-length BIIB antibody (i.e., a single heavy chain and a single light chain); and the third band from the top may be a heavy chain. As shown in FIG.2, there are virtually no degradation products of the full-length BIIB antibody shown on the Western blot. [0084] Densitometry was used to quantify the intensity of each band in the Western blot. Based on the densitometry, the full-length antibody represents 44.5% of all visible antibody bands (the sum of all bands including the full-length antibody). All samples were boiled prior to running on Western blot, making this estimate a lower bound for the amount of full-length antibody secreted by Leishmania tarentolae. The Herceptin reference standard, after boiling and running on the Western blot, showed full-length antibody that represents 68.9% of all visible antibody bands. Similarly, for antibody BIIB, 44.5% is the proportion that remains as a full length after boiling. Therefore, the proportion of full length antibody secreted from the host cell is likely much higher. For example, the host cell may secrete a population of antibodies or antibody fragments in the culture medium, within which at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the population are full-length antibodies. Western Blot Results– Trastuzumab and BIIB antibodies

[0085] The results of the Western blot from Leishmania tarentolae genetically modified to secrete the full-length Herceptin (trastuzumab) are shown in FIG. 4A (see lanes 1-4). The lanes labeled BIIB and Herceptin are standard BIIB and trastuzumab antibodies secreted from CHO cells. 7.5 ul of neat supernant from four separate isolates was run on Western blot analysis against 50 ng of both BIIB standard antibody and Herceptin standard antibody. As shown in FIG. 4A, Leishmania tarentolae secreted the full-length Herceptin antibody, which exhibited a banding pattern indistinguishable from the Herceptin antibody secreted from CHO cells. The top band represents the full-length Herceptin antibody; other bands represent fragments of the full-length antibody. For example, the second band from the top may be a half of the full-length Herceptin antibody (i.e., a single heavy chain and a single light chain); and the third band from the top may be a heavy chain. As shown in FIG. 4A, there are virtually no degradation products of the full-length Herceptin antibody shown on the Western blot. [0086] Densitometry was used to quantify the intensity of each band in the Western blot. Based on the densitometry, full-length trastuzumab antibody represents 50.1% of all visible antibody bands (the sum of all bands including full-length antibody). All samples were boiled prior to running on Western blot, making this estimate a lower bound for the amount of full- length antibody secreted by Leishmania tarentolae. The Herceptin reference standard, after boiling and running the Western, showed full-length antibody represents 67.5% of all visible antibody bands. For the Herceptin antibody, 50.1% is the proportion that remains as a full length after boiling. Therefore, the proportion of full length antibody secreted from the host cell is likely much higher. For example, the host cell may secrete a population of antibodies or antibody fragments in the culture medium, within which at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the population are full-length antibodies. [0087] FIG. 4B illustrates the Western blot analysis of secreted BIIB and Herceptin from L. tarentolae. Production of BIIB antibody was tested in BHI (lane 1 and 2), YE (lane 4 and 5), and YPD (lane 7 and 8) media, all supplemented with 5 μg/mL of hemin. Lane 10, 11, 12, and 13 are samples from four independent clones of L. tarentolae with Herceptin expression cassette. Lane 3, 6, 9, and 14 are supernatant samples from wild type L. tarentolae strain. MW, molecular weight ladder. BIIB and Herceptin are standard antibody samples produced from CHO and 50 ng was loaded on the gels. [0088] As shown in FIG. 4B, L. tarentolae secreted the full-length BIIB and Herceptin antibody, exhibiting a banding pattern indistinguishable from the BIIB and Herceptin antibody secreted from CHO cells. This is the first report of full-length antibody production in L. tarentolae. In BHI media, BIIB antibody titer reached about 4 μg/mL. In the YPD media with porcine hemin added, L. tarentolae produced slightly less than half of the antibody as in the BHI media, which is mainly due to lower biomass achieved in the YPD media. [0089] Expression and secretion IgG monoclonal antibody is a challenge for microbial cells. In addition to the fact that full-length IgG is a large (~150 kDa) and heterotetrameric structure with disulfide linkages, degradation of the full-length antibody is a serious problem. Some examples of degradation problem in fungal systems are described in Kuroda et al., FEMS Yeast Research, 22 August 2007 and Landowski et al., PLoS One, 2017, 10(8): e134723. While it is possible to mitigate degradation by deleting native proteases from the host cell, these manipulations may reduce the production fitness of the host cell and undermine the growth and density advantage of the host cell over other mammalian production systems (e.g., CHO cells). Thus, the fact that Leishmania tarentolae secretes full-length antibodies that display the same banding pattern on Western blot as antibodies secreted from CHO cells indicates that it can be used as a superior antibody production system over CHO cells. Antibody Titer Measurements

[0090] Strains were grown in pre-culture media (BHI, hemin, zeocin, and maintenance antibiotics) for 72 hours as static suspension cultures. Strains were similarly grown in a different culture media, YPD. Strain Y396 has two copies of the secretion signal peptide at the beginning of the antibody sequence and strain Y397 has only one copy. Cells were then sedimented, resuspended in production media (pre-culture media + 100 µg/mL tetracycline) and incubated with continuous agitation. After a 48-hour production period, cells were sedimented and supernatant was run through a 0.2 µm filter. The titer of the full-length BIIB antibody secreted from the host cell into the supernatant was measured using Octet® system (ForteBio). The titer of antibody in the supernatant was at least or about 4 mg per liter. The Octet® titer measurements from two different strains engineered to secrete the full length BIIB antibody are shown in Table 1 below.

Example 2: Glycosylation Pattern of Antibody Produced by Leishmania tarentolae

[0091] Strains were grown in pre-culture media (BHI, hemin, zeocin, and maintenance antibiotics) for 72 hours as static suspension cultures. Cells were then sedimented, resuspended in production media (pre-culture media + 100 µg/mL tetracycline), and incubated with continuous agitation. After a 48-hour production period, cells were sedimented and supernatant was run through a 0.2 µm filter. [0092] The antibody material was purified, and their glycan species were analyzed. As shown in FIG.3, Leishmania tarentolae was able to produce identifiable glycan species. It can produce Man3, which is mammalian-like. [0093] Furthermore, culture broths from Leishmania producing BIIB and Herceptin were Protein A purified and sent to Creative Proteomics for N-glycan profiling analysis. Samples were prepared by Creative Proteomics and analyzed using MALDI-TOF Mass Spectrometer instrument. The data is usually recorded between 500 m/z and 6000 m/z for N-glycans. Results from both antibodies showed a mixture of multiple glycol forms. Among all forms, Man5GlcNAc2 (M5), Gal2GlcNAc2Man3GlcNAc2 (G2), and Gal2GlcNAc2Man3GlcNAc2Fuc1 (G2F) are the most abundant for both antibodies (FIGS.6A and 6B). These forms are produced by human glycosylation pathway and commonly found in therapeutic antibodies. Example 3: Animal Product-Free Culture Media for Leishmania tarentolae and

Optimization of Leishmania tarentolae Growth in Animal Product-Free Media [0094] L. tarentolae is typically grown in Brain Heart Infusion (BHI) media and requires hemin, another animal product, for growth. For pharmaceutical production, fermentations would ideally contain no animal products, and so we explored alternative sources of heme. Catalase purified from Aspergillus niger (Sigma cat # C3515) and peroxidase purified from horseradish (Sigma cat # 77332) were supplied to L. tarentolae cultures in various concentrations to test their effectiveness as non-animal substitutions for hemin in yeast extract (YE) media, with the hypothesis that L. tarentolae can sequester the non-covalently bound heme groups off of these enzymes. L. tarentolae cultures were grown in YE media (1% glucose) supplemented with porcine hemin (0.25% w/v solution in 50% ethanolamine) to a final concentration of 5 µg/mL. Penicillin and streptomycin were added to all media to prevent bacterial contamination. Cultures were diluted (1:10) into buffered yeast extract media with varying concentrations of purified catalase or peroxidase from non-animal sources, and growth was monitored over a 94-hour period. The growth was supported for about 70 hours in this animal product-free and hemin-free culture medium, with catalase supporting faster growth rates and higher cell density than peroxidase (FIGS.5A and 5B). As described herein, in some embodiments, the animal product-free culture media contains peroxidase (e.g., peroxidase purified from horseradish). In some embodiments, the animal product-free culture media contains catalase (e.g., catalase purified from Aspergillus niger). [0095] FIGS.5A and 5B illustrate L. tarentolae growth achieved on animal-free media. FIG. 5A illustrates visualization of average optical density at 600 nm of L. tarentolae growth over a 94 hour period in YE media (1% glucose) supplemented with 0 μg/mL of hemin (solid triangle), 0.5 μg/mL of hemin (open diamond), 200 (solid square), 250 (open square), 300 (solid circle), 350 (open circle), and 400 (solid diamond) μg/mL fungal derived purified catalase enzyme as a substitute for hemin. N=16 wells per condition. FIG. 5B illustrates visualization of average optical density at 600 nm of L. tarentolae growth over a 94 hour period in yeast extract media supplemented with 0 μg/mL of hemin (solid triangle), 0.5 μg/mL of hemin (open diamond), 400 (solid square), 450 (open square), 500 (solid circle), 550 (open circle), and 600 (solid diamond) μg/mL horseradish derived purified peroxidase enzyme as a substitute for hemin. N=16 wells per condition. [0096] In a subsequent experiment, growth of L. tarentolae in shake plates was compared in BHI with hemin, YE media (1% glucose or 1% fructose) with hemin, and YE media (1% glucose or 1% fructose) with 500 μg/mL catalase. At 48 hours, 0.3 μg/mL of hemin or 30 μg/mL of catalase was spiked in the cultures. Growth was followed up to 96 hours. Table 2 summarizes the growth rates and peak cell densities for these culturing conditions. L. tarentolae was able to achieve the same growth rates in YE media with hemin or additional hemin spike as in the BHI media with hemin or hemin spike. Additional hemin spike at 48 hours helped boost peak cell density by 15% compared to no hemin spike in both BHI and YE media. [0097] Reasonable growth rates and peak cell densities were achieved in complete animal- free media. In YE with catalase, growth rate and peak cell density of L. tarentolae were about 57% and 40% of those in BHI or YE media with hemin, respectively. [0098] In certain experiments, instead of diluting the cultures into buffered yeast extract media, the Leishmania cells were separated from supernatant by centrifugation. The supernatant was removed, and the cells were re-suspended in the animal product-free culture medium described above. The growth of the cells was monitored over a 72-hour period. The growth was supported for about 70 hours in this animal product-free (and hemin free) culture medium. [0099] The Leishmania growth rates and cell densities reached in different culture media are summarized in Table 2. Table 2 shows the maximum growth rates and peak cell densities achieved in L. tarentolae cultures while eliminating components of animal origin. At time of inoculation, 5 μg/mL of hemin or 500 μg/mL of catalase was included in the media. At 48 hours, 0.3 μg/mL of hemin or 30 μg/mL of catalase was spiked into the cultures. Growth was followed up to 96 hours. Growth rates measure from 40-48 hours. Table 2: Leishmania growth rates and cell densities (in 96-well plates).

[0100] The data shown in Table 2 indicate that the culture media, where animal product BHI is replaced with yeast extract (YE), can support comparable growth of Leishmania as the culture media containing BHI. See culture media 3) and 4). In culture media 3) and 4) containing yeast extract, the maximum growth rates were slightly better than in culture media 1) and 2) containing BHI. These results indicate that while the commercially available culture media for Leishmania contain animal product (BHI), yeast extracts can replace BHI in a culture medium to support growth for Leishmania. [0101] Hemin is currently available commercially only from an animal source. Since hemin is obtained from an animal source, an alternative source to hemin was sought to replace hemin with non-animal derived hemeprotein so that a culture medium is completely animal product- free to produce full length antibodies. In Table 2, culture media 5), 6), 9), and 10) include catalase containing at least one heme group instead of hemin as a heme source in culture media. The results shown in Table 2 indicate that catalase can replace hemin in a culture media to generate completely animal-free media to support growth for Leishmania. Such completely animal product-free culture media is compatible with production of a therapeutic protein or antibodies for human use. [0102] One or more features from any embodiments described herein or in the figures may be combined with one or more features of any other embodiment described herein in the figures without departing from the scope of the invention. [0103] All publications, patents and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED: 1. A method for producing a genetically engineered non-pathogenic Trypanosomatidae host cell, the method comprising:

(a) contacting a non-pathogenic Trypanosomatidae host cell with a first heterologous nucleic acid sequence encoding an antibody light chain and a second heterologous nucleic acid sequence encoding an antibody heavy chain to produce the genetically engineered non- pathogenic Trypanosomatidae host cell; and

(b) recovering the genetically engineered non-pathogenic Trypanosomatidae host cell, wherein the recovered, genetically engineered non-pathogenic Trypanosomatidae host cell comprises the first and second heterologous nucleic acid sequences in its genome, and is capable of expressing the antibody light chain and the antibody heavy chain and secreting a full-length antibody therefrom, and

wherein the genetically engineered non-pathogenic Trypanosomatidae host cell is capable of secreting a substantially homogenous population of antibodies.

2. The method of claim 1, wherein the first and second heterologous nucleic acid sequences are joined through a linker sequence.

3. The method of claim 2, wherein the linker sequence is a cleavable linker sequence.

4. The method of any one of claims 1 to 3, where the first and second heterologous nucleic acid sequences each comprises a secretion signal sequence.

5. The method of any one of claims 1 to 4, wherein the first and second heterologous nucleic acid sequences are codon optimized according to codon frequencies of the host cell.

6. The method of any one of claims 1 to 5, wherein the host cell is Leishmania tarentolae.

7. The method of any one of claims 1 to 6, wherein the first and second heterologous nucleic acid sequences are in an extrachromosomal expression vector or in an integration vector.

8. The method of any one of claims 1 to 7, wherein at least 50%, 60%, 70%, 80%, 90%, or 95% of the substantially homogenous population of antibodies are full-length antibodies.

9. The method of any one of claims 1 to 8, wherein the full-length antibody is BIIB or trastuzumab.

10. The method of any one of claims 1 to 9, wherein the genetically engineered non- pathogenic Trypanosomatidae host cell is capable of producing at least or about 4 mg/L of the full-length antibody.

11. The method of any one of claims 1 to 10, wherein a low voltage electroporation is applied during contacting the non-pathogenic Trypanosomatidae host cell with the first heterologous nucleic acid sequence and the second heterologous nucleic acid sequence in step (a).

12. The method of any one of claims 1 to 11, wherein the full-length antibody is glycosylated.

13. The method of claim 12, wherein the full-length antibody is mannosylated.

14. A method of producing a full-length antibody, comprising:

(a) providing a non-pathogenic Trypanosomatidae host cell comprising a first heterologous nucleic acid sequence encoding an antibody light chain and a second

heterologous nucleic acid sequence encoding an antibody heavy chain;

(b) culturing the host cell in a cell culture medium comprising a carbon source and a hemeprotein comprising at least one heme group, wherein the cell culture medium is free of animal products;

(c) expressing the antibody light chain and the antibody heavy chain under conditions that allow for the formation of the full-length antibody; and

(d) recovering the full-length antibody,

wherein the method produces a substantially homogenous population of antibodies.

15. The method of claim 14, wherein the non-pathogenic Trypanosomatidae host cell is Leishmania tarentolae.

16. The method of claim 14 or 15, wherein the method produces a cell culture comprising at least or about 4 mg/L of the full-length antibody.

17. The method of any one of claims 14 to 16, wherein the full-length antibody is BIIB or trastuzumab.

18. The method of any one of claims 14 to 17, wherein the cell culture medium comprises yeast extracts.

19. The method of any one of claims 14 to 18, wherein the hemeprotein is a catalase containing at least one heme group.