CROSS-REFERENCE TO RELATED APPLICATIONS

This application incorporates by reference and claims priority to and the benefit of Provisional Patent Application No. 63/315,897, filed on Mar. 2, 2022; Provisional Patent Application No. 63/411,899 filed on Sep. 30, 2022; Provisional Patent Application No. 63/417,873 filed on Oct. 20, 2022, Provisional Patent Application No. 63/436,854 filed on Jan. 3, 2023 and Provisional Patent Application No. 63/448,655 filed on Feb. 27, 2023.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filed electronically in .xml format and is hereby incorporated by reference in its entirety. Said .xml copy, created on Jan. 31, 2023, is Sequence Listing.xml and is 180,965 bytes in size.

FIELD

This application relates to producing recombinant protein in high-productivity cells using serum-free media cultured for large-scale production, with improved systems and methods for measuring and maintaining cell culture conditions, harvesting, and purifying protein having reduced heterogeneity and improved quality with higher yield.

BACKGROUND

Manufacturing processes for a therapeutic protein product should be optimized according to factors including, but not limited to, protein production conditions, protein structure and functional characteristics, and the desired final drug product. Even when using the same therapeutic protein for the same final drug product, changing protein production conditions may be required to optimize titer, batch size, yield, or quality of the product. In turn, the increased and optimized titers, batch sizes and yields require optimizing harvesting and purification processes to process the increased titers and loads while maintaining acceptable quality attributes.

To support increased demand for recombinantly produced protein drug products, cell production processes have been developed that are capable of generating higher titers and improved quality, including reduced heterogeneity and reduced sequence variants, while improving overall yield. Therefore, there is a need for improved cell culture media to meet these demands and specialized cell conditions.

Additionally, measuring and maintaining optimized cell culture conditions are critical and require specially designed equipment, including probes and sensors for measuring dissolved gasses such as oxygen. The use of large-scale vessels and bioreactors, however, can impact the equipment used to measure cell culture conditions, including their accuracy and maintenance requirements. Because the biopharmaceutical industry is highly regulated, irregularities in measurements may require investigation, potentially requiring additional resources and adding delays in manufacturing process. Therefore, there is a need for improved systems and methods for measuring dissolved gasses in large-scale bioreactors that provide improved accuracy and reduce maintenance requirements.

After production, isolating highly concentrated protein presents additional challenges, including limited process volume, filtration capacity, chromatographic column load capacity, and potentially high drug substance viscosity, each of which may affect outcomes such as material use, time spent, product quality, batch purity and batch yield. Thus, novel approaches for harvesting, purifying and formulating high titers of therapeutic protein have also been developed to meet these challenges, at each unit operation of an exemplary harvesting and purification process.

In order to accommodate processing higher titer drug product, improved methods and systems have been developed for manufacturing, harvesting and purifying high titer antibody drug products. For example, the increased titer of drug product and drug loading can exceed the capacity of certain chromatographic purification processes and alter the viscosities which negatively impacts the transfer of drug product along the different chromatography steps. Accordingly, there is a need for improved processes that not only address individual purification steps, but also the entire manufacturing process.

SUMMARY

The present disclosure in part provides an improved process for large scale production of anti-IL4Rα antibody in high-productivity cells. Improved cell culture media and processes for maintaining optimal cell culture conditions have been developed to increase titer and improve quality. Improved processes have also been developed to harvest and purify the increased protein loads while increasing overall yields, reducing manufacturing and operation complexities, and improving overall quality in the drug substance and formulated drug product.

In some embodiments, the anti-IL-4Rα antibody is Dupilumab. In one aspect, the anti-IL-4Rα antibody comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:2. In one aspect, the anti-IL-4Rα antibody comprises three heavy chain complementarity determining region (HCDR) sequences comprising SEQ ID NOs: 3, 4, and 5, and three light chain complementarity determining region (LCDR) sequences comprising SEQ ID NOs: 6, 7, and 8.

In some embodiments the protein of interest is an antigen-binding protein, blocking antibody, or receptor antagonist (e.g., interleukin-4 receptor α (IL-4Rα)). In some aspects, the protein of interest is a protein that has an Fc domain. Thus, in some aspects, the protein of interest is an antibody, such as a human antibody, humanized antibody, chimeric antibody, an antibody fragment, such as a Fab or F(ab′)₂, an ScFv molecule, or the like.

In some embodiments, the manufacturing process includes culturing cells capable of expressing an anti-IL4Rα antibody, or receptor antagonist as described above, in a cell culture media that comprises a base medium that is chemically defined, such as a custom formulation or a commercially available base medium. In another embodiment, the complete media is free of sera (“serum free” media) and/or free of hydrolysate. In another embodiment, the method includes the step of adding one or more point-of-use additions to the cell culture media described below. In some aspects, the point-of-use additions described below can also be included in the cell culture media at the outset.

It has been found that polyamine-supplemented media (“PS media”) increases cell viability and density, reduces cell doubling time, and permits high titer protein production by cells that are grown in such supplemented media. It has been found that PS media provides restored cell viability and density, cell doubling time, and high titer protein production, with or without sera and/or supplemental hydrolysate.

In one embodiment, cells cultured in PS media have an average doubling time that is no more than 30 hours. In one aspect, the cell doubling time is no more than 24 hours. Likewise, the inclusion of polyamines in serum-free media allows cultured cells to reach a higher viable cell density (VCD) than without the inclusion of polyamines.

In one embodiment, the media comprises one or more polyamines selected from the group consisting of ornithine, putrescine, spermine, spermidine or their combinations, in cell culture media that is free of sera and/or free of hydrolysate.

In one embodiment, the PS media is serum-free and comprises ornithine at a concentration of between 30 μM and 900 μM. In one aspect, the ornithine is present in the medium at a concentration (expressed in micromoles per liter) of at least about 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 540, 545, 550, 555, 560, 565, 568, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 620, 625, 630, 635, 640, 645, 650, 700, 750, 800, 850, or 900 μM. Ornithine may be present at between about 0.09 mM and about 0.9 mM. In some embodiments, the PS media comprises ≤7.5 g/L hydrolysate. In some embodiments, the PS media is free of any hydrolysate.

In one embodiment, PS media is serum-free and comprises putrescine at a concentration of between 30 μM and 900 μM. In one aspect the putrescine is present in the medium at a concentration (expressed in micromoles per liter) of at least about 30, 40, 50, 60, 70, 80, 90, 100, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 260, 365, 370, 375, 380, 385, 390, 395, 400, 405, or 410 μM. Putrescine may be present at between about 0.20 mM and about 0.714 mM. In one embodiment, the PS media contains 57 mg/L±8.55 mg/L putrescine·2HCl in addition to ≥15 mg/L±2.25 mg/L ornithine·HCl. In some embodiments, the medium comprises ≤7.5 g/L hydrolysate. In some embodiments, the PS media is free of any hydrolysate.

In one embodiment, the PS media is serum-free and comprises spermine at a concentration of between 10 μM and 900 μM. In one aspect the spermine is present in the medium at a concentration (expressed in micromoles per liter) of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 540, 545, 550, 555, 560, 565, 568, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 620, 625, 630, 635, 640, 645, 650, 700, 750, 800, 850, or 900 μM. In some embodiments, the PS media comprises ≤7.5 g/L hydrolysate. In some embodiments, the PS media is free of any hydrolysate.

In one embodiment, PS media is serum-free and comprises spermidine at a concentration of between 10 μM and 900 μM. In one aspect the spermidine is present in the medium at a concentration (expressed in micromoles per liter) of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 260, 365, 370, 375, 380, 385, 390, 395, 400, 405, or 410 μM. In one embodiment, the PS media contains 57 mg/L±8.55 mg/L spermine·4HCl in addition to ≥15 mg/L±2.25 mg/L spermidine·HCl. In some embodiments, the medium comprises ≤7.5 g/L hydrolysate. In some embodiments, the medium contains ≤16 g/L hydrolysate. In some embodiments, the PS media is free of any hydrolysate.

In one embodiment, the media comprises a mixture of amino acids (with the notable exception of glutamine, which may be added back to the medium as a point of use addition) selected from the group of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and a combination thereof.

In some embodiments, the media comprises at least 40±6 mM or at least 70±10.5 mM of a mixture of amino acids or amino acid salts. In one embodiment, the media comprises at least 40 mM of a mixture of amino acids. In this or another aspect, the media comprises at least 70 mM of a mixture of amino acids. In some embodiments, the total process, including the base medium and feeds, contains a total of at least 115 mM of a mixture of amino acids or amino acid salts.

In some embodiments, the media comprises one or more fatty acids. In one aspect, the medium comprises a mixture of fatty acids (or fatty acid derivatives) and a tocopherol. In one aspect, fatty acids or fatty acid derivatives are selected from the group consisting of linoleic acid, linolenic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, lauric acid, behenic acid, decanoic acid, dodecanoic acid, hexanoic acid, lignoceric acid, myristic acid, and octanoic acid and may can also include thioctic acid derived from fatty acids and a combination thereof.

In some embodiments, the media comprises one or more nucleosides. In one aspect, nucleosides are selected from the group consisting of adenosine, guanosine, cytidine, uridine, thymidine, hypoxanthine, and a combination thereof.

In some embodiments, the cell culture media comprises one or more salts. In some aspects, salts are selected from the group of divalent cations, such as calcium, magnesium, and a combination thereof. In one embodiment, the medium comprises calcium chloride, magnesium sulfate and a combination thereof. In another aspect, salts may also include those of phosphate.

In some embodiments, the cell culture media comprises any one or more of NaHCO₃, glutamine, insulin, glucose, CuSO₄, ZnSO₄, FeCl₃, NiSO₄, Na₄EDTA, Na₃Citrate, and a combination thereof. In one aspect, the method employs the step of adding one or more of the point-of-use chemicals to the cell culture media selected from the group consisting of NaHCO₃, glutamine, insulin, glucose, CuSO₄, ZnSO₄, FeCl₃, NiSO₄, Na₄EDTA, Na₃Citrate, and a combination thereof.

In some embodiments, the point-of-use addition comprises taurine, phosphate, poloxamer 188, and a combination thereof. In some aspects, the point-of-use additions can be included in the medium at the outset.

It has been found that the inclusion of taurine in a cell culture medium increases cellular specific productivity and allows for lower ammonia byproduct by those cells. In one aspect, the cell culture media is serum-free and comprises about 0.1 mM to about 10 mM taurine, about 1 mM to about 9 mM taurine, about 1 mM to about 8 mM taurine, about 1 mM to about 7 mM taurine, about 1 mM to about 6 mM taurine, about 1 mM to about 5 mM taurine, about 1 mM to about 4 mM taurine, about 1 mM to about 3 mM taurine, about 1 mM to about 2 mM taurine, about 0.1 mM to about 1 mM taurine, about 0.2 mM to about 1 mM taurine, about 0.3 mM to about 1 mM taurine, about 0.4 mM to about 1 mM taurine, or about 0.5 mM to about 1 mM taurine.

In some embodiments, the disclosure provides a method for producing an anti-IL-4Rα antibody or antigen binding fragment thereof by employing the steps of (1) introducing into a cell a nucleic acid sequence that encodes a protein of interest; (2) selecting a cell carrying that nucleic acid sequence; (3) culturing the selected cell in embodiments of the serum-free cell culture medium described in this disclosure; and (4) expressing the protein of interest in the cell, wherein the protein of interest is secreted into the medium. The biotherapeutic protein may be a blocking antibody or receptor antagonist (e.g., blocking IL-4Rα and IL-13), and/or an antigen-binding protein, which may comprise an Fc domain. In some embodiments, the antibody is a human, humanized, chimeric or non-human monoclonal antibody or an antibody fragment.

In some embodiments, the cell or cells are mammalian cells, avian cells, insect cells, yeast cells, or bacteria cells. In one aspect, the cells are mammalian cells useful in the production of recombinant proteins, such as CHO cells or the derivative CHO-K1. In some aspects, the cells express a protein of interest, such as a biotherapeutic protein. In some aspects, the cell used in the production of the protein is a mammalian cell capable of producing a biotherapeutic, such as CHO, HEK293, and BHK cell, or any derivatives of them. In one embodiment, the cell is a CHO cell, such as a CHO-K1 cell.

In some embodiments, the cell media (1) is serum-free; (2) comprises hydrolysate; (3) comprises one or more polyamines, including, at least, ornithine or spermine; (4) comprises at least about 40 mM or at least about 70 mM of a mixture of amino acids, including at least one of the following: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and a combination thereof, (5) comprises tocopherol and a mixture of fatty acids; (6) comprises a mixture of nucleosides including at least one of the following: adenosine, guanosine, cytidine, uridine, thymidine, hypoxanthine, and a combination thereof, (7) comprises salts of at least one of the following: calcium, magnesium, and phosphate; and (8) comprises at least one of the following point-of-use additions: NaHCO₃, glutamine, insulin, glucose, CuSO₄, ZnSO₄, FeCl₃, NiSO₄, Na₄EDTA, Na₃citrate, and a combination thereof.

In some embodiments, the cell media (1) is serum-free; (2) comprises one or more polyamines, including, at least, ornithine or spermine; (3) comprises at least about 40 mM or at least about 70 mM of a mixture of amino acids, including at least one of the following: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and a combination thereof, (4) comprises tocopherol and a mixture of fatty acids; (5) comprises a mixture of nucleosides including at least one of the following: adenosine, guanosine, cytidine, uridine, thymidine, hypoxanthine, and a combination thereof, (6) comprises salts, including at least one of the following: calcium, magnesium, phosphate, and a combination thereof, and (7) comprises at least one of the following point-of-use additions: NaHCO₃, glutamine, insulin, glucose, CuSO₄, ZnSO₄, FeCl₃, NiSO₄, Na₄EDTA, Na₃citrate, and a combination thereof.

In some embodiments, the cell media (1) is serum-free; (2) comprises ≤7.5 g/L of a hydrolysate; (3) comprises 0.09±0.014 mM, 0.3±0.05 mM, 0.6±0.09 mM, or 0.9±0.14 mM ornithine; (4) optionally additionally comprises 0.10±0.03 mM, 0.20±0.03 mM, 0.35±0.06 mM, or 0.714±0.11 mM putrescine, spermidine or spermine; (5) comprises at least about 40 mM or at least about 70 mM of a mixture of amino acids including at least one of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and a combination thereof, (6) comprises tocopherol and optionally a mixture of fatty acids; (7) comprises a mixture of nucleosides including adenosine, guanosine, cytidine, uridine, thymidine, hypoxanthine, and a combination thereof; and (8) comprises salts of calcium, magnesium, phosphate, and a combination thereof.

In some embodiments, the cell media (1) comprises no hydrolysate; (2) comprises ornithine at a concentration of at least 90 μM±14 μM; (3) and optionally comprises putrescine, such as at least 150 μM±14 μM. In other embodiments, other polyamines such as spermine, spermidine, and the like are envisaged to be within the scope of the present invention.

In some embodiments, this disclosure provides a method for cultivating cells in a serum-free medium which comprises (1) ornithine at either 0.09±0.014 mM, 0.3±0.05 mM, 0.6±0.09 mM, or 0.9±0.14 mM; (2) optionally additionally putrescine at either 0.20±0.03 mM, 0.35±0.06, or 0.714±0.11 mM; (3) at least about 40 mM or at least about 70 mM of a mixture of amino acids including alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; (4) tocopherol and a mixture of fatty acids; (5) a mixture of nucleosides including adenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine; and (6) salts of calcium, magnesium, and phosphate, wherein the cells cultured according to this method have an average doubling time that is no more than 24 hours or no more than one third the doubling time of cells cultured using similar cell culture medium that comprises less than 0.09±0.015 mM polyamines. In another embodiment, the cell culture is capable of attaining a viable cell density that is at least 3-fold greater than a similar cell culture in a similar cell culture medium that comprises less than 0.09±0.014 mM ornithine (or less than 0.09±0.014 mM ornithine and less than 0.2±0.03 mM putrescine). In one embodiment, the medium comprises ≤7.5 g/L hydrolysate; and in another embodiment, the medium is free of hydrolysates.

In some embodiments, the protein of interest is produced by (1) introducing into a CHO cell a nucleic acid sequence that encodes a protein of interest, such as an antibody or other antigen-binding protein; (2) selecting a cell carrying that nucleic acid sequence; (3) culturing the selected cell in a serum-free cell culture medium which comprises (a) ornithine at either 0.09±0.014, 0.3±0.05 mM, 0.6±0.09 mM, or 0.9±0.14 mM; (b) optionally additionally putrescine at either 0.20±0.03 mM, 0.35±0.06, or 0.714±0.11 mM; (c) at least 40 mM or at least 70 mM of a mixture of amino acids including one or more of: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; (d) tocopherol and optionally a mixture of fatty acids; (e) a mixture of nucleosides including one or more of: adenosine, guanosine, cytidine, uridine, thymidine, hypoxanthine, and a combination thereof, and (f) salts including one or more of: calcium, magnesium, phosphate and a combination thereof; and (4) expressing the protein of interest in the CHO cell, wherein the protein of interest is secreted into the medium. In some embodiments, the serum-free cell culture medium may include ≤7.5 g/L hydrolysates; or in other embodiments no hydrolysates at all.

In some embodiments, the protein of interest is produced by (1) introducing into a CHO cell a nucleic acid sequence that encodes a protein of interest, such as an antibody or other antigen-binding protein; (2) selecting a cell carrying that nucleic acid sequence; (3) culturing the selected cell in a serum-free cell culture medium which comprises (a) ornithine at either 0.09±0.014, 0.3±0.05 mM, 0.6±0.09 mM, or 0.9±0.14 mM; (b) optionally additionally putrescine at either 0.20±0.03 mM, 0.35±0.06, or 0.714±0.11 mM; (c) at least 40 mM or at least 70 mM of a mixture of amino acids including one or more of: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and a combination thereof; (d) tocopherol and a mixture of fatty acids; (e) a mixture of nucleosides including one or more of: adenosine, guanosine, cytidine, uridine, thymidine, hypoxanthine, and a combination thereof, and (f) salts including one or more of: calcium, magnesium, and phosphate; and (4) expressing the protein of interest in the CHO cell, wherein the protein of interest is secreted into the medium. In some embodiments, the serum-free cell culture medium may include ≤7.5 g/L hydrolysates; or in other embodiments no hydrolysates at all.

The present disclosure in part provides an improved seed train process for increasing titer. In one embodiment, the initial viable cell density (VCD) at each step (from N-5 to N-1) is increased to from about 3.5×10⁵to about 5.43×10⁵cells/mL compared to standard initial VCD ranging from 1.7×10⁵to 2.3×10⁵cells/mL. In some aspects, the initial VCD (in N-5 to N-1) is about 1.3×, 1.4×, 1.5×, 1.6, 1.7×, 1.8×, 1.9×, 2.0×, 2.1×, 2.2×, 2.3×, 2.4×, 2.5×, 2.6×, 2.7×, 2.8×, 2.9×, or 3.0× greater than an alternative initial VCD in a standard seed train. In one aspect, the optimized seed train resulted in a 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 1%, 17%, 18%, 19%, or 20% increase in final titer (g/L). In one aspect, there is no substantial difference in peak lactate observed in the final production vessel compared to a standard seed train.

The present disclosure in part provides improved vessels and bioreactors for measuring and controlling cell culturing conditions, including dissolved gasses such as oxygen and carbon dioxide, which are important process parameters for maintenance of a healthy cell culture production process within the biopharmaceutical industry.

The present disclosure in part provides improved methods and systems for air sparging, agitation and measurement of dissolved gasses. More specifically, the present disclosure provides optimized dissolved gas concentrations at various intervals as well as improved bioreactors with optimized agitation rates and working volumes to provide necessary agitation while minimizing shear stress.

In certain exemplary embodiments, the starting volume, air sparge, and agitation setpoints in vessels and bioreactors, including various sensors, along the seed train during the growth phase and production phases are optimized to improve process robustness through minimization of shear stress on the cell culture and optimization of viable cell densities (VCD), dissolved oxygen, carbon dioxide, and pH at various times.

In some embodiments, the sparging rates may be used to vary pCO₂levels within the cell culture to modulate acidic and basic charge variants for cultured cells expressing Dupilumab.

The present disclosure also provides improved methods and systems for measuring dissolved gasses. Electrochemical probes and optical probes may also be used to measure other dissolved gases such as carbon dioxide. Electrochemical probes have been conventionally used in vessels and bioreactors over optical probes because they have a long history of demonstrating a wide linear range and faster response time compared to optical probes or sensors. Electrochemical dissolved oxygen probes, however, require significant upkeep in terms of frequent replacement of membranes and electrolyte solution, and lengthy polarization times. In addition, electrochemical probes are known to provide noisy readings due to bubble interference, with evidence of this occurrence in a large-scale bioreactor provided in the examples below. Such noise can cause excursions outside of normal operating ranges that must be investigated and documented through the quality compliance system of a biopharmaceutical company, due to high regulations within the industry. In the event of short excursions on the high side, generally attributed to bubble interference, an investigation is performed, resulting in strains on manufacturing resources.

Although optical probes are less influenced by bubble interference from agitation, stirring and sparging, and do not have the same electrolyte changing and polarization requirements, optical probes can be less accurate and less responsive compared to electrochemical probes. Further, prior studies assessing the use of optical probes have been limited to water monitoring applications and small-scale cell culture applications. The largest vessel used in the reviewed literature during testing of optical probes was a 50 L single-use bioreactor (Marvell et al., 2009), leaving a gap in the reported scientific literature relating to the use of optical probes in large-scale stainless-steel production (>1,000 L) that have not addressed the performance issues with optical probes.

Accordingly, the present disclosure provides an improved system and method for reducing signal noise in electrochemical probes, which reduces or eliminates false positives attributable to bubble interference. Additionally, the present disclosure provides a system and method for improving accuracy of optical probes used for measuring dissolved gasses in large-scale vessels and bioreactors, which require lower maintenance and are not susceptible to bubble interference compared to conventional electrochemical probes. As discussed in more detail below, vessels and bioreactors with electrochemical probes and optical probes were developed with improved data processing methods to optimize the accuracy of the measurements while minimizing maintenance by reducing or eliminating unwanted excursion events outside of normal operating ranges, which require lengthy quality investigations in the highly regulated biopharmaceutical industry.

In one embodiment, the present disclosure provides methods for measuring VCD of cells cultured in a bioreactor by applying an electric field to cells cultured in a bioreactor, measuring the capacitance, and correlating the measured capacitance to viable cell density.

In one embodiment, the protein of interest is capable of being produced at an average seven-day titer that is at least 7% greater, at least 14% greater, at least 80% greater, at least two-fold greater, or at least three-fold greater than the average seven-day titer produced by a similar cell in a serum-free cell culture medium that comprises less than 0.09±0.014 mM ornithine (or less than 0.09±0.014 mM ornithine and less than 0.2±0.03 mM putrescine).

To harvest and purify the increased protein production, new and improved methods were developed that not only can accommodate the increased protein production but improve on the overall yield percentage. The methods include, for example, optimizations of harvest pre-treatment, protein A chromatography, viral inactivation, anion exchange chromatography, cation exchange chromatography, hydrophobic interaction chromatography, virus retentive filtration, concentration and diafiltration, and drug substance adjustment, which are further described below.

This disclosure provides a method for purifying an anti-IL4Rα antibody. In some exemplary embodiments, the method comprises (a) subjecting said antibody to pre-treatment; (b) harvesting said antibody; (c) subjecting said antibody to affinity chromatography; (d) subjecting antibody pooled from eluate in step (c) to viral inactivation at a pH from about 3 to about 4.5 and then adjusting the pH to from about 5 to about 8; (e) subjecting said antibody pooled from step (d) to anion exchange chromatography in flowthrough mode; (f) subjecting said antibody pooled from flowthrough fractions of step (e) to cation exchange chromatography in bind and elute mode; (g) subjecting said antibody pooled from eluate of step (f) to hydrophobic interaction chromatography in flowthrough mode; and (h) subjecting said antibody pooled from flowthrough fractions of step (g) to virus retentive filtration.

In one aspect, the antibody of step (h) is further subjected to concentration and diafiltration using a diafiltration buffer having a pH between 4.0 and 4.5.

In one aspect, the diafiltration buffer comprises from about 4 mM acetate to about 6 mM acetate.

In one aspect, harvest pre-treatment includes subjecting said antibody to transient pH levels from about 4.5 to 5.0 or about 4.0 to about 5.5 and then increasing the pH levels to from about 5.5 to 6.5.

In one aspect, the affinity chromatography is Protein A chromatography.

In one aspect, the harvesting of step (b) includes centrifugation and depth filtration.

In one aspect, the antibody is harvested in a 3 kL to 25 kL bioreactor.

In one aspect, the Protein A column load pH is between 7 and 8. In one aspect, the Protein A column load pH is between 6 and 8. In one aspect, the Protein A column load pH is about 6.

In alternate exemplary embodiments, the method comprises (a) culturing cells expressing the anti-IL-4Rα antibody or an antigen-binding fragment thereof, (b) subjecting said cells to transient pH levels from about 4.5 to 5.0 or about 4.0 to about 5.5, then adjusting the pH to from about 5.5 to 6.5; (c) harvesting said cells by centrifugation to separate cell debris from clarified media comprising the anti-IL-4Rα antibody or antigen-binding fragment thereof, (d) subjecting said clarified media to affinity chromatography; (e), subjecting the anti-IL-4Rα antibody or antigen-binding fragment thereof pooled from eluate in step (d) to viral inactivation at a pH from about 3 to about 4.5 and then adjusting the pH to from about 5 to about 8; (f) subjecting the anti-IL-4Rα antibody or antigen-binding fragment thereof pooled from step (e) to anion exchange chromatography in flowthrough mode; (g) subjecting the anti-IL-4Rα antibody or antigen-binding fragment thereof pooled from flowthrough fractions of step (f) to cation exchange chromatography in bind and elute mode; (h) subjecting the anti-IL-4Rα antibody or antigen-binding fragment thereof pooled from eluate of step (g) to hydrophobic interaction chromatography in flowthrough mode; and (i) subjecting the anti-IL-4Rα antibody or antigen-binding fragment thereof pooled from flowthrough fractions of step (h) to virus retentive filtration, thereby producing the anti-IL-4Rα antibody or antigen-binding fragment thereof.

In one aspect, the method further comprises subjecting said anti-IL-4Rα antibody or antigen-binding fragment to ultrafiltration and diafiltration (UF/DF) after step (i).

In one aspect, the affinity chromatography is Protein A chromatography.

In one aspect, the anti-IL-4Rα antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining region (HCDR) sequences comprising SEQ ID NOs: 3, 4, and 5, and three light chain complementarity determining region (LCDR) sequences comprising SEQ ID NOs: 6, 7, and 8.

In one aspect, the anti-IL-4Rα antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 2.

In one aspect, the anti-IL-4Rα antibody is Dupilumab.

These and other aspects of the present disclosure will be better appreciated and understood when considered in conjunction with the following description and accompanying drawings. The following description, while indicating various embodiments and numerous specific details thereof, is given by way of illustration and not of limitation. Various substitutions, modifications, additions, or rearrangements may be made within the scope of the invention, and additional Enumerated Examples are provided below describing additional aspects of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the structure of an antibody product, Dupilumab, according to an exemplary embodiment.

FIG. 2 represents the effect of insulin supplementation on titer profile of Dupilumab, according to an exemplary embodiment.

FIG. 3 represents the effect of insulin supplementation on viability of cells expressing Dupilumab, according to an exemplary embodiment.

FIG. 4 shows the effect of insulin supplementation on the reduction of ammonia levels in the culture comprising cells expressing Dupilumab, according to an exemplary embodiment.

FIG. 5 shows the effect of insulin concentration of 15 mg/L and feed schedule on growth rate of the titer curve, upper asymptote (highest achievable titer), inflection point, and titer, according to an exemplary embodiment.

FIG. 6 shows the effect of insulin supplementation using the profiler for day 13 on high molecular weight species, according to an exemplary embodiment.

FIG. 7 shows the effect of insulin supplementation using the profiler for day 13 on levels of non-reduced purity, according to an exemplary embodiment.

FIG. 8 shows the effect of insulin supplementation schedule using the profiler for day 13 on non-glycosylated heavy chain (NGHC) levels, according to an exemplary embodiment.

FIG. 9 shows the effect of tyrosine supplementation on titer and ammonia levels in a cell culture expressing Dupilumab, according to an exemplary embodiment.

FIG. 10 shows the effect of phosphate redistribution strategy 6 and 8 on Dupilumab titer (g/L), according to an exemplary embodiment.

FIG. 11 shows phosphate redistribution profiles on days 0, 2, 4, 6, and 8, according to an exemplary embodiment.

FIG. 12A shows a contour plot of central composite design (CCD) model predicting host cell protein clearance factor (CF), according to an exemplary embodiment.

FIG. 12B shows a contour plot of CCD model predicting normalized supernatant turbidity (NTU), according to an exemplary embodiment.

FIG. 13A shows a contour plot of CCD model predicting host cell protein clearance factor, according to an exemplary embodiment.

FIG. 13B shows a contour plot of CCD model predicting normalized supernatant turbidity (NTU), according to an exemplary embodiment.

FIG. 14 shows the initial and final viable cell density (VCD) for an average standard seed train and an optimized seed train with an increased initial VCD in comparison to a standard seed train, according to an exemplary embodiment.

FIG. 15 shows the biomass decline in a 10 kL production bioreactor for cells cultured using an optimized seed train with an increased initial VCD in comparison to a standard seed train, according to an exemplary embodiment.

FIG. 16A and FIG. 16B show the final titer (g/L) of an optimized seed train (Sample 1) with an increased initial VCD in comparison to standard seed trains (Samples 2 to 9), according to an exemplary embodiment.

FIG. 17 shows a bivariate linear regression of the on-line capacitance measurements and off-line VCD measurements of cell line A (CLA) during seed train, according to an exemplary embodiment of the present invention.

FIG. 18 shows bivariate linear regressions of the on-line capacitance measurements and off-line VCD measurements of CLA during seed train N-3, N-2 and N-1 phases, according to an exemplary embodiment of the present invention.

FIG. 19 shows predictions of the on-line VCD measurements of CLA during seed train N-3, N-2 and N-1 phases which were determined using the on-line capacitance measurements and the correlation equation presented in FIG. 21, according to an exemplary embodiment of the present invention. The dots (circles) indicate VCD values determined off-line using a bioanalyzer.

FIG. 20 shows a bivariate linear regression of the on-line capacitance measurements and off-line VCD measurements of cell line B (CLB) during seed train, according to an exemplary embodiment of the present invention.

FIG. 21 shows bivariate linear regressions of the on-line capacitance measurements and off-line VCD measurements of CLB during seed train N-3, N-2 and N-1 phases, according to an exemplary embodiment of the present invention.

FIG. 22 shows predictions of the on-line VCD measurements of CLB during seed train N-3, N-2 and N-1 phases, which were determined using the on-line capacitance measurements and the correlation equation presented in FIG. 24, according to an exemplary embodiment of the present invention. The dots (circles) indicate VCD values determined off-line using a bioanalyzer.

FIG. 23 shows a bivariate linear regression of the on-line capacitance measurements and off-line VCD measurements of CLA and CLB during seed train, according to an exemplary embodiment of the present invention.

FIG. 24A, FIG. 24B, and FIG. 24C show predictions of the on-line VCD measurements of CLA during seed train N-3, N-2 and N-1 phases, which were determined using the on-line capacitance measurements presented in FIG. 17 and the correlation equations presented in FIG. 17 (FIG. 24A), FIG. 20 (FIG. 24B) and FIG. 23 (FIG. 24C), according to an exemplary embodiment of the present invention. The dots (circles) indicate VCD values determined off-line using a bioanalyzer.

FIG. 25A, FIG. 25B, and FIG. 25C show predictions of the on-line VCD measurements of CLB during seed train N-3, N-2 and N-1 phases (black trace), which were determined using the on-line capacitance measurements presented in FIG. 21 and the correlation equations presented in FIG. 20 (FIG. 25A), FIG. 17 (FIG. 25B) and FIG. 23 (FIG. 25C), according to an exemplary embodiment of the present invention. The dots (circles) indicate VCD values determined off-line using a bioanalyzer.

FIG. 26A and FIG. 26B show predictions of the on-line VCD measurements of CLB during seed train and production phases, which were determined using on-line capacitance measurements and the correlation equation presented in FIG. 20, according to an exemplary embodiment of the present invention. Panels A and B show the data for two separate cultures, each of which used a single probe to perform on-line capacitance measurements. In FIG. 26A, the line depicts the predictions of the on-line VCD measurements and the dots (circles) indicate VCD values determined off-line using a bioanalyzer. In FIG. 26B, the line depicts the predictions of the on-line VCD measurements and the dots (circles) indicate VCD values determined off-line using a bioanalyzer.

FIG. 27A shows the host cell protein values and Dupilumab yield post-acid precipitation of Dupilumab cell culture fluid using 1M phosphoric acid, according to an exemplary embodiment.

FIG. 27B shows the host cell protein values and Dupilumab yield post-acid precipitation of Dupilumab cell culture fluid using 1.75 M acetic acid, according to an exemplary embodiment.

FIG. 28 shows a schematic of an exemplary outline regarding the use of depth filters, polish filter and guard filters, according to an exemplary embodiment.

FIG. 29 shows a prediction profiler modeling the factors and response studied in the centrifuge characterization study, according to an exemplary embodiment.

FIG. 30 shows a schematic of exemplary purification steps of the optimized process, according to an exemplary embodiment.

FIG. 31 shows a schematic of exemplary purification steps of the optimized process, according to an exemplary embodiment.

FIG. 32 shows a visualization of models generated for a multivariate study of affinity capture and viral inactivation risk factors and responses, according to an exemplary embodiment.

FIG. 33 shows HCP levels in affinity capture pool when using various affinity wash buffers, according to an exemplary embodiment.

FIG. 34 shows HCP levels following AEX or HIC processing downstream of affinity capture chromatography using various affinity wash buffers, according to an exemplary embodiment.

FIG. 35 shows protein yield and HMW species in affinity capture pool when using various affinity wash buffers, according to an exemplary embodiment.

FIG. 36 shows a visualization of models generated for a multivariate study of AEX risk factors and responses, according to an exemplary embodiment.

FIG. 37 shows a visualization of models generated for a multivariate study of CEX risk factors and responses, according to an exemplary embodiment.

FIG. 38 shows a visualization of models generated for a multivariate study of HIC risk factors and responses, according to an exemplary embodiment.

FIG. 39 shows a visualization of models generated for a multivariate study of VRF risk factors and responses, according to an exemplary embodiment.

FIG. 40 shows a visualization of models generated for a multivariate study of UF/DF risk factors and responses, according to an exemplary embodiment.

FIG. 41 shows bioreactor titer for Dupilumab 500 L confirmation batches compared to 10,000 L process performance qualification batches and 2 L mirror productions, according to an exemplary embodiment.

FIG. 42 shows step yield by unit operation for 500 L confirmation batches compared to 10,000 L process performance qualification batches, according to an exemplary embodiment.

FIG. 43 shows HMW content by unit operation for 500 L confirmation batches compared to 10,000 L process performance qualification batches, according to an exemplary embodiment.

FIG. 44 shows HCP content by unit operation for 500 L confirmation batches compared to 10,000 L process performance qualification batches, according to an exemplary embodiment.

FIG. 45 illustrates an example of a stepped agitation and air sparging rate over time, according to an exemplary embodiment.

FIG. 46 illustrates an example of signal filtering for cell culture with a 59 second sampling rate and ˜10-minute sample window applying an Agile filter, according to an exemplary embodiment. The label “A” illustrates that excursions on the low side remain for the smoothed signal. The label “B” illustrates good agreement between smoothed and unfiltered signals for expected process disturbances.

FIG. 47 illustrates an example of signal filtering for cell culture with a 59 second sampling rate and ˜33-minute sample window, applying an Agile filter, according to an exemplary embodiment. The label “A” illustrates that excursions on the low side are removed for the smoothed signal. The label “B” illustrates good agreement between smoothed and unfiltered signals for expected process disturbances.

FIG. 48 illustrates an example of signal filtering for cell culture with a 59 second sampling rate and ˜33-minute sample window, applying a Savitzky-Golay filter, according to an exemplary embodiment. The label “A” illustrates that noise remains for the filtered signal. The label “B” illustrates good agreement between smoothed and unfiltered signals for expected process disturbances.

FIG. 49 illustrates a stacked comparison of the filtered signal from FIG. 46-FIG. 48, according to an exemplary embodiment.

FIG. 50 illustrates Run 1 dissolved oxygen trends (where the light shaded line is Electrochemical, medium shaded line is Optical Probe B, darkest shaded line is Optical Probe A, Dashed lines: setpoint and normal operating range upper and lower limits), according to an exemplary embodiment.

FIG. 51 illustrates Run 2 dissolved oxygen trends (where the light shaded line is Electrochemical, medium shaded line is Optical Probe B, darkest shaded line is Optical Probe A, Dashed lines: setpoint and normal operating range upper and lower limits), according to an exemplary embodiment.

FIG. 52 illustrates Run 3 dissolved oxygen trends (where the light shaded line is Electrochemical, medium shaded line is Optical Probe B, darkest shaded line is Optical Probe A, Dashed lines: setpoint and normal operating range upper and lower limits), according to an exemplary embodiment.

FIG. 53 illustrates Run 4 dissolved oxygen trends (where the light shaded line is Electrochemical, medium shaded line is Optical Probe B, darkest shaded line is Optical Probe A, Dashed lines: setpoint and normal operating range upper and lower limits), according to an exemplary embodiment.

FIG. 54 illustrates dissolved oxygen data generated using Optical Probe B with an anti-bubble cap for Runs 1-3 and a standard cap for Run 4, according to an exemplary embodiment.

FIG. 55 illustrates dissolved oxygen data as determined at time segments at ‘Start’, ‘Middle’ and ‘End’ of Run 2 examined as part of the offset assessment (where the light shaded line is Electrochemical, medium shaded line is Optical Probe B, darkest shaded line is Optical Probe A, the bottom most line is Electrochemical with Agile filter (59 s sampling rate and default sample window)), according to an exemplary embodiment.

FIG. 56 illustrates Run 1 Probe B Offset correction (where the medium shaded line is Optical Probe B, the darkest shaded line is Optical Probe B adjusted upwards by 3.0%, the light shaded line is Electrochemical with Agile filter (59 s sampling rate and default sample window), Dashed lines: setpoint and normal operating range upper/lower limits), according to an exemplary embodiment.

FIG. 57 illustrates Run 3 Probe B Offset Correction (where the medium shaded line is Optical Probe B, the darkest shaded line is Optical Probe B adjusted upwards by 3.0%, the light shaded line is Electrochemical with Agile filter (59 s sampling rate and default sample window), Dashed lines: setpoint and normal operating range upper/lower limits), according to an exemplary embodiment.

FIG. 58A and FIG. 58B illustrate examples of varying dextrose concentrations at various times.

FIG. 59 illustrates pCO₂profiles of culture media with varying sparging conditions in mmHg (y-axis) over process time (days), according to an exemplary embodiment. Medium pCO2 was selected as a mid-point control.

FIG. 60 shows a visualization of models generated for a multivariate study of mixed-mode chromatography (MMC) risk factors and responses using Capto Adhere resin, according to an exemplary embodiment.

FIG. 61 shows a visualization of models generated for a multivariate study of MMC risk factors and responses using PPA HyperCel resin, according to an exemplary embodiment.

DETAILED DESCRIPTION

Manufacturing processes for a therapeutic protein product, for example a monoclonal antibody drug, should be optimized according to factors including, but not limited to, recombinant host cell characteristics, protein production conditions, protein structure and functional characteristics, and the desired final drug product. To support increased demand for recombinantly produced protein drug products, cell lines and cell culture media have been developed that are capable of generating higher titers and larger batch sizes, with improved quality and yield.

Even when using the same therapeutic protein for the same final drug product, changing protein production conditions may be required to optimize titer, batch size, yield, or quality of the product. The applicants have made the discovery that the addition of polyamines, such as one or a combination of ornithine, putrescine, spermine and spermidine, to a medium (“PS media”) improves viable cell density, cell doubling time, and protein production of Dupilumab by a cell in a cell culture relative to a serum-free medium that comprises very little or no polyamines such as ornithine, putrescine, spermine and spermidine (“non-PS media”).

Conventional methods for harvesting and purifying protein drug products rely on a series of filtering and chromatography steps that may be limited in terms of their capacity to process increased titers and corresponding batch size, protein concentrations and viscosity of drug product using similarly sized equipment, while maintaining purity and quality standards. For example, pumps previously used to move drug product may fail when moving drug product with higher protein concentrations, due to increased viscosity levels. Techniques have been developed to reduce sample viscosity, for example using the addition of arginine, a hydrophobic salt. However, adding arginine during harvesting and purification, particularly during concentration and diafiltration, leads to the loss of unknown quantities of arginine in later processing steps, which must then be measured and compensated for further downstream, and may add variability to the final drug product. Therefore, a need exists for systems and methods for harvesting a high titer of antibody drug product while maintaining product consistency, high yield and quality.

The disclosure herein provides improvements to manufacturing, harvesting and purification processes for improving the production of a high titer antibody product. In some exemplary embodiments, a high titer antibody product of interest is Dupilumab.

Dupilumab is a human monoclonal immunoglobulin G4 (IgG4) antibody expressed and prepared from Chinese hamster ovary cells (CHO-K1) targeting interleukin-4 receptor subunit a (IL-4R-α). After cell cultivation, harvesting, isolation and purification, it is prepared for administration with, for example, histidine, histidine hydrochloride, arginine hydrochloride, polysorbate 80, sodium acetate, glacial acetic acid, sucrose and water.

Dupilumab inhibits interleukin-4 (IL-4) and interleukin-13 (IL-13) signaling by specifically binding to the IL-4 receptor α (IL-4Rα) subunit shared by the IL-4 and IL-13 receptor complexes. Dupilumab inhibits IL-4 signaling via the Type I receptor and both IL-4 and IL-13 signaling through the Type II receptor. Blocking IL4Rα with Dupilumab inhibits IL-4 and IL-13 cytokine-induced inflammatory responses, including the release of proinflammatory cytokines, chemokines, nitric oxide, and IgE. By blocking the obligate shared component of the IL-4/IL-13 receptor complex, Dupilumab inhibits IL-4 and IL-13 signaling, key disease drivers of Type II inflammation, and provides clinical benefit and long-term disease control without the side effects commonly observed with existing non-selective systemic immunosuppressants.

Dupilumab received its first worldwide marketing approval for the treatment of moderate-to-severe atopic dermatitis (AD) in adults in the United States on 28 Mar. 2017, then in the European Union on 28 Sep. 2017, in Japan on 19 Jan. 2018, and subsequently in multiple other countries. Since then, Dupilumab has been approved for treatment of other diseases associated with Type II inflammation, including moderate-to-severe asthma, chronic rhinosinusitis with nasal polyposis (CRSwNP), eosinophilic esophagitis (EoE), chronic spontaneous urticaria (CSU), and prurigo nodularis (PN). In addition, a subcutaneous administration of Dupilumab is currently being developed for the treatment of patients with type 2 inflammatory diseases, including AD (pediatric patients (6 months to <6 years)), asthma (pediatric patients (6 months to <6 years)), chronic obstructive pulmonary disease (COPD), allergic fungal rhino-sinusitis (AFRS), chronic rhinosinusitis without nasal polyps (CRSsNP), allergies (e.g. grass allergy, peanut allergy, and dairy allergy), bullous pemphigoid, hand and foot atopic dermatitis, cold-induced urticaria, chronic inducible urticaria, ulcerative colitis, chronic pruritis of unknown origin, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis, bronchiectasis, alopecia areata, and allergic rhinitis.

In order to meet a high demand for Dupilumab, a novel manufacturing process was developed, as described further below. The novel manufacturing process allows for, for example, the production and purification of Dupilumab 150 mg/mL and 175 mg/mL formulated drug substance (FDS) at 10,000 L scale and 25,000 L scale.

Description of Terms

Unless described otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein known to the skilled artisan can be used in the practice of particular embodiments described herein. All publications mentioned are hereby incorporated by reference in their entirety.

The term “a” should be understood to mean “at least one” and the terms “about” and “approximately” should be understood to permit standard variation as would be understood by those of ordinary skill in the art, and where ranges are provided, endpoints are included. As used herein, the terms “include,” “includes,” and “including” are meant to be non-limiting and are understood to mean “comprise,” “comprises,” and “comprising” respectively.

As used herein, the term “upstream process technology,” in the context of protein preparation, refers to activities involving the production of proteins from cells in a cell culture. As used herein, the term “cell culture” refers to methods for generating and maintaining a population of host cells capable of producing a recombinant protein of interest, as well as the methods and techniques for optimizing the production and collection of the protein of interest. For example, once an expression vector has been incorporated into an appropriate host cell, the host cell can be maintained under conditions suitable for expression of the relevant nucleotide coding sequences, and the collection and production of the desired recombinant protein.

The present disclosure provides a serum-free medium that is useful in culturing cells and producing a biopharmaceutical drug substance. “Serum-free” applies to a cell culture medium that does not comprise animal sera, such as fetal bovine serum. The serum-free media may comprise ≤7.5 g/L of hydrolysates, such as soy hydrolysate. The present disclosure also provides chemically defined media (CDM), which is not only serum-free, but also hydrolysate-free.

“Hydrolysate-free” applies to cell culture media that comprises no exogenous protein hydrolysates, such as animal or plant protein hydrolysates, including, for example, peptones, tryptones and the like.

“Cell culture” or “culture” means the growth and propagation of cells outside of a multicellular organism or tissue. Suitable culture conditions for mammalian cells are known in the art. See e.g., Animal cell culture: A Practical Approach, D. Rickwood, ed., Oxford University Press, New York (1992). Mammalian cells may be cultured in suspension or while attached to a solid substrate. Fluidized bed bioreactors, hollow fiber bioreactors, roller bottles, flasks, or stirred tank bioreactors, with or without microcarriers, and operated in a batch, fed batch, continuous, semi-continuous, or perfusion mode are available for mammalian cell culture. Cell culture media or concentrated feed media may be added to the culture continuously or at intervals during the culture. For example, a culture may be fed once per day, every other day, every three days, or may be fed when the concentration of a specific medium component, which is being monitored, falls outside a desired range.

As used herein, the term “chemically defined medium” or “chemically defined media” (both abbreviated “CDM”) refers to a synthetic growth medium in which the identity and concentration of all the ingredients are defined. Chemically defined media do not comprise bacterial, yeast, animal, or plant extracts, animal serum, or plasma, although individual plant or animal-derived components (e.g., proteins, polypeptides, etc.) may be added. Chemically defined media may comprise inorganic salts such as phosphates, sulfates, and the like needed to support growth. The carbon source is defined, and is usually a sugar such as glucose, lactose, galactose, and the like, or other compounds such as glycerol, lactate, acetate, and the like. While certain chemically defined culture media also use phosphate salts as a buffer, other buffers may be employed such as sodium bicarbonate, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), citrate, triethanolamine, and the like. Examples of commercially available chemically defined media include, but are not limited to, various Dulbecco's Modified Eagle's (DME) media (Sigma-Aldrich Co; SAFC Biosciences, Inc.), Ham's Nutrient Mixture (Sigma-Aldrich Co; SAFC Biosciences, Inc.), various EX-CELLs mediums (Sigma-Aldrich Co; SAFC Biosciences, Inc.), various IS CHO-CD mediums (FUJIFILM Irvine Scientific), combinations thereof, and the like. Methods of preparing chemically defined culture media are known in the art, for example, in U.S. Pat. Nos. 6,171,825 and 6,936,441, WO 2007/077217, and U.S. Patent Application Publication Nos. 2008/0009040 and 2007/0212770, the entire teachings of which are herein incorporated by reference.

As used herein, the term “recombinant host cell” (or simply “host cell”) includes a cell into which a recombinant expression vector coding for a protein of interest has been introduced. It should be understood that such a term is intended to refer not only to a particular subject cell but to a progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. In an embodiment, host cells include prokaryotic and eukaryotic cells selected from any of the kingdoms of life. In one aspect, eukaryotic cells include protist, fungal, plant and animal cells. In a further aspect, host cells include eukaryotic cells such as plant and/or animal cells. The cells can be mammalian cells, fish cells, insect cells, amphibian cells or avian cells. In a particular aspect, the host cell is a mammalian cell. A wide variety of mammalian cell lines suitable for growth in culture are available from the American Type Culture Collection (Manassas, Va.) and other depositories as well as commercial vendors. Cells that can be used in the processes of the invention include, but are not limited to, MK2.7 cells, PER-C6 cells, Chinese hamster ovary cells (CHO), such as CHO-K1 (ATCC CCL-61), (Chasin et al., 1986, Som. Cell Molec. Genet., 12:555-556; Kolkekar et al., 1997, Biochemistry, 36: 10901-10909; and WO 01/92337 A2), dihydrofolate reductase negative CHO cells (CHO/-DHFR, Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA, 77:4216), and dp12.CHO cells (U.S. Pat. No. 5,721,121); monkey kidney cells (CV1, ATCC CCL-70); monkey kidney CV1 cells transformed by SV40 (COS cells, COS-7, ATCC CRL-1651); HEK293 cells, Sp2/0 cells, 5L8 hybridoma cells, Daudi cells, EL4 cells, HeLa cells, HL-60 cells, K562 cells, Jurkat cells, THP-1 cells, Sp2/0 cells, primary epithelial cells (e.g., keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, kidney epithelial cells and retinal epithelial cells) and established cell lines and their strains (e.g., human embryonic kidney cells (e.g., HEK293 cells, or HEK293 cells subcloned for growth in suspension culture, Graham et al., 1977, J. Gen. Virol., 36:59); baby hamster kidney cells (BHK, ATCC CCL-10); mouse sertoli cells (TM4, Mather, 1980, Biol. Reprod., 23:243-251); human cervical carcinoma cells (HELA, ATCC CCL-2); canine kidney cells (MDCK, ATCC CCL-34); human lung cells (W138, ATCC CCL-75); human hepatoma cells (HEP-G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL-51); buffalo rat liver cells (BRL 3A, ATCC CRL-1442); TRI cells (Mather, 1982, Annals NY Acad. Sci., 383:44-68); MCR 5 cells; FS4 cells; PER-C6 retinal cells, MDBK (NBL-1) cells, 911 cells, CRFK cells, MDCK cells, BeWo cells, Chang cells, Detroit 562 cells, HeLa 229 cells, HeLa S3 cells, Hep-2 cells, KB cells, LS 180 cells, LS 174T cells, NCI-H-548 cells, RPMI 2650 cells, SW-13 cells, T24 cells, WI-28 VA13, 2RA cells, WISH cells, BS-C-I cells, LLC-MK₂cells, Clone M-3 cells, 1-10 cells, RAG cells, TCMK-1 cells, Y-1 cells, LLC-PK₁cells, PK(15) cells, GH₁cells, GH₃cells, L2 cells, LLC-RC 256 cells, MH₁C₁cells, XC cells, MDOK cells, VSW cells, and TH-I, B1 cells, or derivatives thereof), fibroblast cells from any tissue or organ (including but not limited to heart, liver, kidney, colon, intestines, esophagus, stomach, neural tissue (brain, spinal cord), lung, vascular tissue (artery, vein, capillary), lymphoid tissue (lymph gland, adenoid, tonsil, bone marrow, and blood), spleen, and fibroblast and fibroblast-like cell lines (e.g., TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells, citrullinemia cells, Dempsey cells, Detroit 551 cells, Detroit 510 cells, Detroit 525 cells, Detroit 529 cells, Detroit 532 cells, Detroit 539 cells, Detroit 548 cells, Detroit 573 cells, HEL 299 cells, IN4R-90 cells, MRC-5 cells, WI-38 cells, WI-26 cells, MiCl₁cells, CV-1 cells, COS-1 cells, COS-3 cells, COS-7 cells, African green monkey kidney cells (VERO-76, ATCC CRL-1587; VERO, ATCC CCL-81); DBS-FrhL-2 cells, BALB/3T3 cells, F9 cells, SV-T2 cells, M-MSV-BALB/3T3 cells, K-BALB cells, BLO-11 cells, NOR-10 cells, C₃H/IOTI/2 cells, HSDM₁C₃cells, KLN205 cells, McCoy cells, Mouse L cells, Strain 2071 (Mouse L) cells, L-M strain (Mouse L) cells, L-MTK (Mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss/3T3 cells, Indian muntac cells, SIRC cells, C_IIcells, and Jensen cells, or derivatives thereof) or any other cell type known to one skilled in the art. In some aspects, cells used in the invention are CHO cells, HEK293 cells, BHK cells, or derivatives thereof.

As used herein, the term “host cell proteins” (HCP) includes protein derived from a host cell and can be unrelated to the desired protein of interest. Host cell proteins can be a process-related impurity which can be derived from the manufacturing process and can include three major categories: cell substrate-derived, cell culture-derived and downstream derived. Cell substrate-derived impurities include, but are not limited to, proteins derived from a host organism and nucleic acid (host cell genomic, vector, or total DNA). Cell culture-derived impurities include, but are not limited to, inducers, antibiotics, serum, and other media components. Downstream-derived impurities include, but are not limited to, enzymes, chemical and biochemical processing reagents (e.g., cyanogen bromide, guanidine, oxidizing and reducing agents), inorganic salts (e.g., heavy metals, arsenic, nonmetallic ion), solvents, carriers, ligands (e.g., monoclonal antibodies), and other leachables.

As used herein, the term “liquid chromatography” refers to a process in which a biological/chemical mixture carried by a liquid can be separated into components as a result of differential distribution of the components as they flow through (or into) a stationary liquid or solid phase. Non-limiting examples of liquid chromatography include reverse phase liquid chromatography, ion-exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, or mixed-mode chromatography. In some aspects, a sample comprising the at least one protein of interest or peptide digest can be subjected to any one of the aforementioned chromatographic methods or a combination thereof. Analytes separated using chromatography will feature distinctive retention times, reflecting the speed at which an analyte moves through the chromatographic column. Analytes may be compared using a chromatogram, which plots retention time on one axis and measured signal on another axis, where the measured signal may be produced from, for example, UV detection or fluorescence detection.

As used herein, the term “mass spectrometer” includes a device capable of identifying specific molecular species and measuring their accurate masses. The term is meant to include any molecular detector with which a polypeptide or peptide may be characterized. A mass spectrometer can include three major parts: the ion source, the mass analyzer, and the detector. The role of the ion source is to create gas phase ions. Analyte atoms, molecules, or clusters can be transferred into gas phase and ionized either concurrently (as in electrospray ionization) or through separate processes. The choice of ion source depends on the application.

In some exemplary embodiments, the mass spectrometer can be a tandem mass spectrometer. As used herein, the term “tandem mass spectrometry” includes a technique where structural information on sample molecules is obtained by using multiple stages of mass selection and mass separation. A prerequisite is that the sample molecules be transformed into a gas phase and ionized so that fragments are formed in a predictable and controllable fashion after the first mass selection step. MS/MS, or MS², can be performed by first selecting and isolating a precursor ion (MS¹), and fragmenting it to obtain meaningful information. Tandem MS has been successfully performed with a wide variety of analyzer combinations. Which analyzers to combine for a certain application can be determined by several different factors, such as sensitivity, selectivity, and speed, but also size, cost, and availability. The two major categories of tandem MS methods are tandem-in-space and tandem-in-time, but there are also hybrids where tandem-in-time analyzers are coupled in space or with tandem-in-space analyzers. A tandem-in-space mass spectrometer comprises an ion source, a precursor ion activation device, and at least two non-trapping mass analyzers. Specific m/z separation functions can be designed so that in one section of the instrument ions are selected, dissociated in an intermediate region, and the product ions are then transmitted to another analyzer for m/z separation and data acquisition. In tandem-in-time, mass spectrometer ions produced in the ion source can be trapped, isolated, fragmented, and m/z separated in the same physical device.

The peptides identified by the mass spectrometer can be used as surrogate representatives of the intact protein and their post-translational modifications or other modifications. They can be used for protein characterization by correlating experimental and theoretical MS/MS data, the latter generated from possible peptides in a protein sequence database. The characterization includes, but is not limited, to sequencing amino acids of the protein fragments, determining protein sequencing, determining protein de novo sequencing, locating post-translational modifications, or identifying post-translational modifications, or comparability analysis, or combinations thereof.

In some exemplary aspects, the mass spectrometer can work on nanoelectrospray or nanospray. The term “nanoelectrospray” or “nanospray” as used herein refers to electrospray ionization at a very low solvent flow rate, typically hundreds of nanoliters per minute of sample solution or lower, often without the use of an external solvent delivery. The electrospray infusion setup forming a nanoelectrospray can use a static nanoelectrospray emitter or a dynamic nanoelectrospray emitter. A static nanoelectrospray emitter performs a continuous analysis of small sample (analyte) solution volumes over an extended period of time. A dynamic nanoelectrospray emitter uses a capillary column and a solvent delivery system to perform chromatographic separations on mixtures prior to analysis by the mass spectrometer.

In some exemplary embodiments, mass spectrometry can be performed under native conditions. As used herein, the term “native conditions” can include performing mass spectrometry under conditions that preserve non-covalent interactions in an analyte. For a detailed review on native MS, refer to the review: Elisabetta Boeri Erba & Carlo Petosa, The emerging role of native mass spectrometry in characterizing the structure and dynamics of macromolecular complexes, 24 PROTEIN SCIENCE 1176-1192 (2015).

As used herein, the term “database” refers to a compiled collection of protein sequences that may possibly exist in a sample, for example in the form of a file in a FASTA format. Relevant protein sequences may be derived from cDNA sequences of a species being studied. Public databases that may be used to search for relevant protein sequences included databases hosted by, for example, Uniprot or Swiss-prot. Databases may be searched using what are herein referred to as “bioinformatics tools.” Bioinformatics tools provide the capacity to search uninterpreted MS/MS spectra against all possible sequences in the database(s) and provide interpreted (annotated) MS/MS spectra as an output. Non-limiting examples of such tools are Mascot (www.matrixscience.com), Spectrum Mill (www.chem.agilent.com), PLGS (www.waters.com), PEAKS (www.bioinformaticssolutions.com), Proteinpilot (download.appliedbiosystems.com/proteinpilot), Phenyx (www.phenyx-ms.com), Sorcerer (www.sagenresearch.com), OMSSA (www.pubchem.ncbi.nlm.nih.gov/omssa/), X!Tandem (www.thegpm.org/TANDEMI), Protein Prospector (prospector.ucsf.edu/prospector/mshome.htm), Byonic (www.proteinmetrics.com/products/byonic) or Sequest (fields.scripps.edu/sequest).

As used herein, the term “downstream process technology” refers to one or more techniques used after the upstream process technologies to produce, process, and/or purify a protein. Downstream process technology includes, for example, isolation of a protein product, using, for example, affinity chromatography, ion exchange chromatography, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, or displacement chromatography.

As used herein, the term “ultrafiltration” or “UF” can include a membrane filtration process similar to reverse osmosis, using hydrostatic pressure to force water through a semi-permeable membrane. Ultrafiltration is described in detail in: LEOS J. ZEMAN & ANDREW L. ZYDNEY, MICROFILTRATION AND ULTRAFILTRATION: PRINCIPLES AND APPLICATIONS (1996), the entire teaching of which is herein incorporated. Filters with a pore size of smaller than 0.1 μm can be used for ultrafiltration. By employing filters having such small pore size, the volume of the sample can be reduced through permeation of the sample buffer through the filter while proteins are retained behind the filter.

As used herein, “diafiltration” or “DF” can include a method of using ultrafilters to remove and exchange salts, sugars, and non-aqueous solvents, to separate free from bound species, to remove low molecular weight material, and/or to cause the change of ionic and/or pH environments. Microsolutes are removed most efficiently by adding solvent to a solution being ultrafiltered at a rate approximately equal to the ultrafiltration rate. This washes microspecies from the solution at a constant volume. In certain exemplary embodiments of the present disclosure, a diafiltration step can be employed to exchange various buffers used in connection with the instant disclosure, for example, prior to chromatography or other production steps, as well as to remove impurities from the protein preparation.

As used herein, the term “formulation” refers to a pharmaceutical product, for example Dupilumab, that is formulated together with one or more pharmaceutically acceptable vehicles.

In terms of protein formulation, the term “stable,” as used herein refers to the protein of interest within the formulation being able to retain an acceptable degree of chemical structure or biological function after storage under exemplary conditions defined herein. A formulation may be stable even though the protein of interest contained therein does not maintain 100% of its chemical structure or biological function after storage for a defined amount of time. Under certain circumstances, maintenance of about 90%, about 95%, about 96%, about 97%, about 98% or about 99% of a protein's structure or function after storage for a defined amount of time may be regarded as “stable.”

The term “treat” or “treatment” refers to a therapeutic measure that reverses, stabilizes or eliminates an undesired disease or disorder (e.g., atopic dermatitis (eczema), eosinophilic or oral steroid dependent asthma, chronic rhinosinusitis with nasal polyposis (CRSwNP), eosinophilic esophagitis (EoE), inflammation), for example, by causing the regression, stabilization or elimination of one or more symptoms or indicia of such disease or disorder by any clinically measurable degree, for example, with regard to asthma it helps prevent severe asthma attacks (exacerbations), can improve breathing, and helps reduce the amount of oral corticosteroids needed.

As used herein, the term “critical quality attribute” (CQA) is used to describe a physical, chemical, biological, or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality. CQAs may include, for example, post-translational modifications.

As used herein, “viral filtration” can include filtration using suitable filters including, but not limited to, Planova 20N™, 50 N or BioEx from Asahi Kasei Pharma, Viresolve™ filters from EMD Millipore, ViroSart® CPV or Virosart® HF from Sartorius, or Ultipor DV20 or DV50™ or Pegasus™ Prime filter from Pall Corporation. It will be apparent to one of ordinary skill in the art to select a suitable filter to obtain desired filtration performance.

For biologics, the implementation of a robust and flexible upstream process is desirable. An efficient upstream process can lead to desirable production and scale-up of a protein of interest.

As used herein, a “sample” can be obtained from any step of a bioprocess, such as cell culture fluid (CCF), harvested cell culture fluid (HCCF), any step in the downstream processing, drug substance (DS), or a drug product (DP) comprising the final formulated product. In some specific exemplary embodiments, the sample can be selected from any step of the downstream process of clarification, chromatographic production, or filtration.

As used herein, the term “protein alkylating agent” or “alkylation agent” refers to an agent used for alkylating certain free amino acid residues in a protein. Non-limiting examples of protein alkylating agents are iodoacetamide (IOA/IAA), chloroacetamide (CAA), acrylamide (AA), N-ethylmaleimide (NEM), methyl methanethiosulfonate (MMTS), and 4-vinylpyridine or combinations thereof.

As used herein, “protein denaturing” or “denaturation” can refer to a process in which the three-dimensional shape of a molecule is changed from its native state. Protein denaturation can be carried out using a protein denaturing agent. Non-limiting examples of a protein denaturing agent include heat, high or low pH, reducing agents like DTT, SDS or exposure to chaotropic agents. Several chaotropic agents can be used as protein denaturing agents. Chaotropic solutes increase the entropy of the system by interfering with intramolecular interactions mediated by non-covalent forces such as hydrogen bonds, van der Waals forces, and hydrophobic effects. Non-limiting examples of chaotropic agents include butanol, ethanol, guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate (SDS), thiourea, N-lauroylsarcosine, urea, and salts thereof. In some exemplary embodiments, denaturing agents may be used in the mobile phase in a chromatography analysis. In some exemplary embodiments, a denaturing agent used in a mobile phase may be acetonitrile (ACN). In some exemplary embodiments, a denaturing agent used in a mobile phase may be a surfactant.

As used herein, the term “digestion” refers to hydrolysis of one or more peptide bonds of a protein. There are several approaches to carrying out digestion of a protein in a sample using an appropriate hydrolyzing agent, for example, enzymatic digestion or non-enzymatic digestion. Digestion of a protein into constituent peptides can produce a “peptide digest” that can further be analyzed using peptide mapping analysis.

As used herein, the term “hydrolyzing agent” refers to any one or combination of a large number of different agents that can perform digestion of a protein. Non-limiting examples of hydrolyzing agents that can carry out enzymatic digestion include protease from Aspergillus saitoi, elastase, subtilisin, protease XIII, pepsin, trypsin, Tryp-N, chymotrypsin, aspergillopepsin I, LysN protease (Lys-N), LysC endoproteinase (Lys-C), endoproteinase Asp-N (Asp-N), endoproteinase Arg-C (Arg-C), endoproteinase Glu-C (Glu-C) or outer membrane protein T (OmpT), immunoglobulin-degrading enzyme of Streptococcus pyogenes (IdeS), thermolysin, papain, pronase, V8 protease or biologically active fragments or homologs thereof or combinations thereof. Non-limiting examples of hydrolyzing agents that can carry out non-enzymatic digestion include the use of high temperature, microwave, ultrasound, high pressure, infrared, solvents (non-limiting examples are ethanol and acetonitrile), immobilized enzyme digestion (IMER), magnetic particle immobilized enzymes, and on-chip immobilized enzymes. For a recent review discussing the available techniques for protein digestion, see Switzar et al., “Protein Digestion: An Overview of the Available Techniques and Recent Developments” (Linda Switzar, Martin Giera & Wilfried M. A. Niessen, Protein Digestion: An Overview of the Available Techniques and Recent Developments, 12 Journal of Proteome Research 1067-1077 (2013), the entire teachings of which are herein incorporated). One or a combination of hydrolyzing agents can cleave peptide bonds in a protein or polypeptide, in a sequence-specific manner, generating a predictable collection of shorter peptides. The ratio of hydrolyzing agent to protein and the time required for digestion can be appropriately selected to obtain optimal digestion of the protein. When the enzyme to substrate ratio is unsuitably high, the correspondingly high digestion rate will not allow sufficient time for the peptides to be analyzed by mass spectrometer, and sequence coverage will be compromised. On the other hand, a low E/S ratio would need long digestion and thus long data acquisition time. The enzyme to substrate ratio can range from about 1:0.5 to about 1:200.

As used herein, the term “protein reducing agent” or “reduction agent” refers to the agent used for reduction of disulfide bridges in a protein. Non-limiting examples of protein reducing agents used to reduce a protein are dithiothreitol (DTT), 8-mercaptoethanol, Ellman's reagent, hydroxylamine hydrochloride, sodium cyanoborohydride, tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl), or combinations thereof.

As used herein, the term “protein” or “protein of interest” can include any amino acid polymer having covalently linked amide bonds. Proteins comprise one or more amino acid polymer chains, generally known in the art as “polypeptides.” “Polypeptide” refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds. “Synthetic peptide or polypeptide” refers to a non-naturally occurring peptide or polypeptide. Synthetic peptides or polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. Various solid phase peptide synthesis methods are known to those of skill in the art. A protein may comprise one or multiple polypeptides to form a single functioning biomolecule. In another exemplary aspect, a protein can include antibody fragments, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, and the like. Proteins of interest can include any of bio-therapeutic proteins, recombinant proteins used in research or therapy, chimeric proteins, antibodies, monoclonal antibodies, polyclonal antibodies, and human antibodies. Proteins may be produced using recombinant cell-based production systems, such as the insect baculovirus system, yeast systems (e.g., Pichia sp.), and mammalian systems (e.g., CHO cells and CHO derivatives like CHO-K1 cells). For a recent review discussing biotherapeutic proteins and their production, see Ghaderi et al., “Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation” (Darius Ghaderi et al., Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation, 28 BIOTECHNOLOGY AND GENETIC ENGINEERING REVIEWS 147-176 (2012), the entire teachings of which are herein incorporated by reference). In some exemplary embodiments, proteins comprise modifications, adducts, and other covalently linked moieties. These modifications, adducts and moieties include, for example, avidin, streptavidin, biotin, glycans (e.g., N-acetylgalactosamine, galactose, neuraminic acid, N-acetylglucosamine, fucose, mannose, and other monosaccharides), PEG, polyhistidine, FLAGtag, maltose binding protein (MBP), chitin binding protein (CBP), glutathione-S-transferase (GST), myc-epitope, fluorescent labels and other dyes, and the like. Proteins can be classified on the basis of compositions and solubility and can thus include simple proteins, such as globular proteins and fibrous proteins; conjugated proteins, such as nucleoproteins, glycoproteins, mucoproteins, chromoproteins, phosphoproteins, metalloproteins, and lipoproteins; and derived proteins, such as primary derived proteins and secondary derived proteins.

As used herein, the term “recombinant protein” refers to a protein produced as the result of the transcription and translation of a gene carried on a recombinant expression vector that has been introduced into a suitable host cell. In certain exemplary embodiments, the recombinant protein can be an antibody, for example, a chimeric, humanized, or fully human antibody. In certain exemplary embodiments, the recombinant protein can be an antibody of an isotype selected from group consisting of. IgG, IgM, IgA1, IgA2, IgD, and IgE. In certain exemplary embodiments the antibody molecule is a full-length antibody (e.g., an IgG1) or alternatively the antibody can be a fragment (e.g., an Fc fragment or a Fab fragment).

The term “antibody” as used herein includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In different embodiments of the present disclosure, the FRs of the anti-big-ET-1 antibody (or antigen-binding portion thereof) may be identical to the human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs. The term “antibody,” as used herein, also includes antigen-binding fragments of full antibody molecules. The terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, for example, from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and/or is readily available from, for example, commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

As used herein, an “antibody fragment” includes a portion of an intact antibody, such as, for example, the antigen-binding or variable region of an antibody. Examples of antibody fragments include, but are not limited to, a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment, a scFv fragment, a Fv fragment, a dsFv diabody, a dAb fragment, a Fd′ fragment, a Fd fragment, and an isolated complementarity determining region (CDR) region, as well as triabodies, tetrabodies, linear antibodies, and single-chain antibody molecules. Fv fragments are the combination of the variable regions of the immunoglobulin heavy and light chains, and ScFv proteins are recombinant single chain polypeptide molecules in which immunoglobulin light and heavy chain variable regions are connected by a peptide linker. In some exemplary embodiments, an antibody fragment comprises a sufficient amino acid sequence of the parent antibody of which it is a fragment that it binds to the same antigen as does the parent antibody; in some exemplary embodiments, a fragment binds to the antigen with a comparable affinity to that of the parent antibody and/or competes with the parent antibody for binding to the antigen. An antibody fragment may be produced by any means. For example, an antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody and/or it may be recombinantly produced from a gene encoding the partial antibody sequence. Alternatively, or additionally, an antibody fragment may be wholly or partially synthetically produced. An antibody fragment may optionally comprise a single chain antibody fragment. Alternatively, or additionally, an antibody fragment may comprise multiple chains that are linked together, for example, by disulfide linkages. An antibody fragment may optionally comprise a multi-molecular complex. A functional antibody fragment typically comprises at least about 50 amino acids and more typically comprises at least about 200 amino acids.

The term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. A monoclonal antibody can be derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, by any means available or known in the art. Monoclonal antibodies useful with the present disclosure can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.

As used herein, a “protein pharmaceutical product,” “biopharmaceutical product” or “biotherapeutic” includes an active ingredient which can be fully or partially biological in nature. In one aspect, the protein pharmaceutical product can comprise a peptide, a protein, an antibody, an antigen, a peptide-drug conjugate, an antibody-drug conjugate, a protein-drug conjugate, cells, tissues, or combinations thereof. In another aspect, the protein pharmaceutical product can comprise a recombinant, engineered, modified, mutated, or truncated version of a peptide, a protein, an antibody, an antigen, vaccine, a peptide-drug conjugate, an antibody-drug conjugate, a protein-drug conjugate, cells, tissues, or combinations thereof.

Exemplary Anti-IL-4Rα Antibodies

As used herein, Dupilumab (IgG4 isotype) is a covalent heterotetramer consisting of two disulfide-linked human heavy chains, each covalently linked through a disulfide bond to a human kappa light chain. Each heavy chain comprises a serine-to-proline mutation at amino acid 233, which is located in the hinge region of the Fc domain, to reduce the propensity of the antibody to form half-antibodies in solution. There is a single N-linked glycosylation site (Asn³⁰²) in each heavy chain, located within the CH2 domain of the Fc constant region in the molecule. The antibody, based on the primary sequence (in the absence of N-linked glycosylation), possesses a molecular weight of 146,897.0 Da, taking into account the formation of 16 disulfide bonds and removal of Lys⁴⁵²from each heavy chain C-terminus. The variable domains of the heavy and light chains combine to form complementarity-determining regions (CDRs) for the binding of Dupilumab to its target, interleukin-4 receptor α (IL-4Rα). A schematic representation of Dupilumab, including the location of N-linked glycosylation sites and disulfide bond structures, is presented in FIG. 1.

The manufacturing process can be used to produce a human antibody or antigen-binding fragment thereof that specifically binds IL-4R (such as IL-4Rα) and comprises the three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) having an amino acid sequence of SEQ ID NO: 1. The antibody or antigen-binding fragment may comprise the three light chain CDRs (LCVR1, LCVR2, LCVR3) contained within a light chain variable region (LCVR) having an amino acid sequence of SEQ ID NO: 2. Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and/or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify the boundaries of CDRs include, e.g., the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of the structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within an antibody.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises the six CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3) from the heavy and light chain variable region amino acid sequence pairs (HCVR/LCVR) of SEQ ID NOs: 1 and 2.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises six CDRs (HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3) having the amino acid sequences of SEQ ID NOs: 3/4/5/6/7/8.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 1 and 2.

In certain embodiments, the antibody is Dupilumab, which comprises the HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 1 and 2.

In certain embodiments, the antibody sequence is Dupilumab, which comprises the heavy chain/light chain amino acid sequence pair of SEQ ID NOs: 9 and 10.

Dupilumab HCVR Amino Acid Sequence:

(SEQ ID NO: 1)

EVQLVESGGGLEQPGGSLRLSCAGSGFTFRDYAMTWVRQAPGKGLEWVS

SISGSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK

DRLSITIRPRYYGLDVWGQGTTVTVS.

Dupilumab LCVR Amino Acid Sequence:

(SEQ ID NO: 2)

DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSIGYNYLDWYLQKSGQSP

QLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGFYYCMQALQ

TPYTFGQGTKLEIK.

Dupilumab HCDR1 Amino Acid Sequence:

(SEQ ID NO: 3)

GFTFRDYA.

Dupilumab HCDR2 Amino Acid Sequence:

(SEQ ID NO: 4)

ISGSGGNT.

Dupilumab HCDR3 Amino Acid Sequence:

(SEQ ID NO: 5)

AKDRLSITIRPRYYGL.

Dupilumab LCDR1 Amino Acid Sequence:

(SEQ ID NO: 6)

QSLLYSIGYNY.

Dupilumab LCDR2 Amino Acid Sequence:

(SEQ ID NO: 7)

LGS.

Dupilumab LCDR3 Amino Acid Sequence:

(SEQ ID NO: 8)

MQALQTPYT.

Dupilumab HC Amino Acid Sequence:

(SEQ ID NO: 9)

EVQLVESGGGLEQPGGSLRLSCAGSGFTFRDYAMTWVRQAPGKGLEWVS

SISGSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK

DRLSITIRPRYYGLDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSEST

AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV

PSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT

KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK

AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP

ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY

TQKSLSLSLG (amino acids 1-124 = HCVR; amino acids

125-451 = HC constant).

Dupilumab LC Amino Acid Sequence:

(SEQ ID NO: 10)

DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSIGYNYLDWYLQKSGQSP

QLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGFYYCMQALQ

TPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR

EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV

YACEVTHQGLSSPVTKSFNRGEC (amino acids 1-112 =

LCVR; amino acids 112-219 = LC constant).

In certain embodiments, the manufacturing method can be used to produce an antibody or antigen-binding fragment thereof comprising light chain variable region (LCVR) and heavy chain variable region (HCVR) sequence pairs (LCVR/HCVR) selected from the group consisting of SCB-VL-39/SCB-VH-92; SCB-VL-40/SCB-VH-92; SCB-VL-41/SCB-VH-92; SCB-VL-42/SCB-VH-92; SCB-VL-43/SCB-VH-92; SCB-VL-44/SCB-VH-92; SCB-VL-44/SCB-VH-62; SCB-VL-44/SCB-VH-68; SCB-VL-44/SCB-VH-72; SCB-VL-44/SCB-VH-82; SCB-VL-44/SCB-VH-85; SCB-VL-44/SCB-VH-91; SCB-VL-44/SCB-VH-93; SCB-VL-45/SCB-VH-92; SCB-VL-46/SCB-VH-92; SCB-VL-47/SCB-VH-92; SCB-VL-48/SCB-VH-92; SCB-VL-49/SCB-VH-92; SCB-VL-50/SCB-VH-92; SCB-VL-51/SCB-VH-92; SCB-VL-51/SCB-VH-93; SCB-VL-52/SCB-VH-92; SCB-VL-52/SCB-VH-62; SCB-VL-52/SCB-VH-91; SCB-VL-53/SCB-VH-92; SCB-VL-54/SCB-VH-92; SCB-VL-54/SCB-VH-62; SCB-VL-54/SCB-VH-68; SCB-VL-54/SCB-VH-72; SCB-VL-54/SCB-VH-82; SCB-VL-54/SCB-VH-85; SCB-VL-54/SCB-VH-91; SCB-VL-55/SCB-VH-92; SCB-VL-55/SCB-VH-62; SCB-VL-55/SCB-VH-68; SCB-VL-55/SCB-VH-72; SCB-VL-55/SCB-VH-82; SCB-VL-55/SCB-VH-85; SCB-VL-55/SCB-VH-91; SCB-VL-56/SCB-VH-92; SCB-VL-57/SCB-VH-92; SCB-VL-57/SCB-VH-93; SCB-VL-57/SCB-VH-59; SCB-VL-57/SCB-VH-60; SCB-VL-57/SCB-VH-61; SCB-VL-57/SCB-VH-62; SCB-VL-57/SCB-VH-63; SCB-VL-57/SCB-VH-64; SCB-VL-57/SCB-VH-65; SCB-VL-57/SCB-VH-66; SCB-VL-57/SCB-VH-67; SCB-VL-57/SCB-VH-68; SCB-VL-57/SCB-VH-69; SCB-VL-57/SCB-VH-70; SCB-VL-57/SCB-VH-71; SCB-VL-57/SCB-VH-72; SCB-VL-57/SCB-VH-73; SCB-VL-57/SCB-VH-74; SCB-VL-57/SCB-VH-75; SCB-VL-57/SCB-VH-76; SCB-VL-57/SCB-VH-77; SCB-VL-57/SCB-VH-78; SCB-VL-57/SCB-VH-79; SCB-VL-57/SCB-VH-80; SCB-VL-57/SCB-VH-81; SCB-VL-57/SCB-VH-82; SCB-VL-57/SCB-VH-83; SCB-VL-57/SCB-VH-84; SCB-VL-57/SCB-VH-85; SCB-VL-57/SCB-VH-86; SCB-VL-57/SCB-VH-87; SCB-VL-57/SCB-VH-88; SCB-VL-57/SCB-VH-89; SCB-VL-57/SCB-VH-90; SCB-VL-57/SCB-VH-91; SCB-VL-58/SCB-VH-91; SCB-VL-58/SCB-VH-92; and SCB-VL-58/SCB-VH-93.

In certain embodiments, an antibody or antigen-binding fragment thereof comprises a LCVR/HCVR sequence pair of SCB-VL-44/SCB-VH-92.

In certain embodiments, an antibody or antigen-binding fragment thereof comprises a LCVR/HCVR sequence pair of SCB-VL-54/SCB-VH-92.

In certain embodiments, an antibody or antigen-binding fragment thereof comprises a LCVR/HCVR sequence pair of SCB-VL-55/SCB-VH-92.

In certain embodiments, an antibody or antigen-binding fragment thereof comprises an HCVR comprising an HCDR1 sequence of SCB-92-HCDR1, an HCDR2 sequence of SCB-92-HCDR2, and an HCDR3 sequence of SCB-92-HCDR3, and an LCVR comprising an LCDR1 sequence of SCB-55-LCDR1, an LCDR2 sequence of SCB-55-LCDR2, and an LCDR3 sequence of SCB-55-LCDR3.

The antibodies recited below in Table 1 are described in more detail in U.S. Pat. No. 10,774,141, incorporated herein by reference in its entirety for all purposes.

TABLE 1

Sequence ID
Sequence

SCB-VL-39
EIVLTQSPGTLSLSPGERATLSCRASQSVSNSYLAWYQQKPGQAPRLL

IFGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPP

WTFGQGTKVEIK (SEQ ID NO: 11)

SCB-VL-40
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLL

IYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPP

WTFGQGTKVEIK (SEQ ID NO: 12)

SCB-VL-41
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLL

IFGASSRAPGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPP

WTFGQGTKVEIK (SEQ ID NO: 13)

SCB-VL-42
EIVLTQSPGTLSLSPGERATLSCRASQSVSNSYLAWYQQKPGQAPRLL

IYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPP

WTFGQGTKVEIK (SEQ ID NO: 14)

SCB-VL-43
EIVLTQSPGTLSLSPGERATLSCRASQSVSNSYLAWYQQKPGQAPRLL

IFGASSRAPGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPP

WTFGQGTKVEIK (SEQ ID NO: 15)

SCB-VL-44
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLL

IYGASSRAPGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPP

WTFGQGTKVEIK (SEQ ID NO: 16)

SCB-VL-45
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLL

IFGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDHSPP

WTFGQGTKVEIK (SEQ ID NO: 17)

SCB-VL-46
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLL

IFGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSAG

WTFGQGTKVEIK (SEQ ID NO: 18)

SCB-VL-47
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLL

IFGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDHSAG

WTFGQGTKVEIK (SEQ ID NO: 19)

SCB-VL-48
EIVLTQSPGTLSLSPGERATLSCRASQSVSNSYLAWYQQKPGQAPRLL

IFGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDHSPP

WTFGQGTKVEIK (SEQ ID NO: 20)

SCB-VL-49
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLL

IYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDHSPP

WTFGQGTKVEIK (SEQ ID NO: 21)

SCB-VL-50
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLL

IFGASSRAPGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDHSPP

WTFGQGTKVEIK (SEQ ID NO: 22)

SCB-VL-51
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLL

IYGASSRAPGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDHSA

GWTFGQGTKVEIK (SEQ ID NO: 23)

SCB-VL-52
EIVLTQSPGTLSLSPGERATLSCRASQSVSNSYLAWYQQKPGQAPRLL

IFGASSRAPGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDHSAG

WTFGQGTKVEIK (SEQ ID NO: 24)

SCB-VL-53
EIVLTQSPGTLSLSPGERATLSCRASQSVSNSYLAWYQQKPGQAPRLL

IYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDHSA

GWTFGQGTKVEIK (SEQ ID NO: 25)

SCB-VL-54
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLL

IFGASSRAPGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDHSAG

WTFGQGTKVEIK (SEQ ID NO: 26)

SCB-VL-55
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLL

IYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDHSA

GWTFGQGTKVEIK (SEQ ID NO: 27)

SCB-VL-56
EIVLTQSPGTLSLSPGERATLSCRASQSVSNSYLAWYQQKPGQAPRLL

IFGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDHSAG

WTFGQGTKVEIK (SEQ ID NO: 28)

SCB-VL-57
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLL

IFGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPP

WTFGQGTKVEIK (SEQ ID NO: 29)

SCB-VL-58
EIVLTQSPGTLSLSPGERATLSCRASQSVSNSYLAWYQQKPGQAPRLL

IYGASSRAPGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDHSA

GWTFGQGTKVEIK (SEQ ID NO: 30)

SCB-VH-59
EVQLVESGGGLVHPGGSLRLSCAGSGFTFSRNAMFWVRQAPGKGLE

WVSGIGTGGATNYADSVKGRFTISRDNAKNSLYLQMNSLRAEDMA

VYYCARGRYYFDYWGQGTLVTVSS (SEQ ID NO: 31)

SCB-VH-60
EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSRNAMFWVRQAPGKGLE

WVSGIGTGGATNYADSVKGRFTISRDNAKNSLYLQMNSLRAEDMA

VYYCARGRYYFDYWGQGTLVTVSS (SEQ ID NO: 32)

SCB-VH-61
EVQLVQSGGGLVHPGGSLRLSCAASGFTFSRNAMFWVRQAPGKGLE

WVSGIGTGGATNYADSVKGRFTISRDNAKNSLYLQMNSLRAEDMA

VYYCARGRYYFDYWGQGTLVTVSS (SEQ ID NO: 33)

SCB-VH-62
EVQLVQSGGGLVHPGGSLRLSCAGSGFTFSRNAMFWVRQAPGKGLE

WVSGIGTGGATSYADSVKGRFTISRDNAKNSLYLQMNSLRAEDMAV

YYCARGRYYFDYWGQGTLVTVSS (SEQ ID NO: 34)

SCB-VH-63
EVQLVQSGGGLVHPGGSLRLSCAGSGFTFSRNAMFWVRQAPGKGLE

WVSGIGTGGATNYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV

YYCARGRYYFDYWGQGTLVTVSS (SEQ ID NO: 35)

SCB-VH-64
EVQLVESGGGLVQPGGSLRLSCAGSGFTFSRNAMFWVRQAPGKGLE

WVSGIGTGGATNYADSVKGRFTISRDNAKNSLYLQMNSLRAEDMA

VYYCARGRYYFDYWGQGTLVTVSS (SEQ ID NO: 36)

SCB-VH-65
EVQLVESGGGLVHPGGSLRLSCAASGFTFSRNAMFWVRQAPGKGLE

WVSGIGTGGATNYADSVKGRFTISRDNAKNSLYLQMNSLRAEDMA

VYYCARGRYYFDYWGQGTLVTVSS (SEQ ID NO: 37)

SCB-VH-66
EVQLVQSGGGLVQPGGSLRLSCAASGFTFSRNAMFWVRQAPGKGLE

WVSGIGTGGATNYADSVKGRFTISRDNAKNSLYLQMNSLRAEDMA

VYYCARGRYYFDYWGQGTLVTVSS (SEQ ID NO: 38)

SCB-VH-67
EVQLVQSGGGLVHPGGSLRLSCAGSGFTFSRNAMFWVRQAPGKGLE

WVSGIGTGGATSYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV

YYCARGRYYFDYWGQGTLVTVSS (SEQ ID NO: 39)

SCB-VH-68
EVQLVQSGGGLVHPGGSLRLSCAGSGFTFSRNAMFWVRQAPGKGLE

WVSGIGTGGATNYADSVKGRFTISRDNAKNSLYLQMNSLRAEDMA

VYYCARGRYYFPWWGQGTLVTVSS (SEQ ID NO: 40)

SCB-VH-69
EVQLVESGGGLVHPGGSLRLSCAGSGFTFSRNAMFWVRQAPGKGLE

WVSGIGTGGATNYADSVKGRFTISRDNAKNSLYLQMNSLRAEDMA

VYYCARGRYYFPWWGQGTLVTVSS (SEQ ID NO: 41)

SCB-VH-70
EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSRNAMFWVRQAPGKGLE

WVSGIGTGGATNYADSVKGRFTISRDNAKNSLYLQMNSLRAEDMA

VYYCARGRYYFPWWGQGTLVTVSS (SEQ ID NO: 42)

SCB-VH-71
EVQLVQSGGGLVHPGGSLRLSCAASGFTFSRNAMFWVRQAPGKGLE

WVSGIGTGGATNYADSVKGRFTISRDNAKNSLYLQMNSLRAEDMA

VYYCARGRYYFPWWGQGTLVTVSS (SEQ ID NO: 43)

SCB-VH-72
EVQLVQSGGGLVHPGGSLRLSCAGSGFTFSRNAMFWVRQAPGKGLE

WVSGIGTGGATSYADSVKGRFTISRDNAKNSLYLQMNSLRAEDMAV

YYCARGRYYFPWWGQGTLVTVSS (SEQ ID NO: 44)

SCB-VH-73
EVQLVQSGGGLVHPGGSLRLSCAGSGFTFSRNAMFWVRQAPGKGLE

WVSGIGTGGATNYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV

YYCARGRYYFPWWGQGTLVTVSS (SEQ ID NO: 45)

SCB-VH-74
EVQLVQSGGGLVHPGRSLRLSCAGSGFTFSRNAMFWVRQAPGKGLE

WVSGIGTGGATNYADSVKGRFTISRDNAKNSLYLQMNSLRAEDMA

VYYCARGRYYFDYWGQGTLVTVSS (SEQ ID NO: 46)

SCB-VH-75
EVQLVQSGGGLVHPGGSLRLTCAGSGFTFSRNAMFWVRQAPGKGLE

WVSGIGTGGATNYADSVKGRFTISRDNAKNSLYLQMNSLRAEDMA

VYYCARGRYYFDYWGQGTLVTVSS (SEQ ID NO: 47)

SCB-VH-76
EVQLVQSGGGLVHPGGSLRLSCAGSGFTFSRNAMHWVRQAPGKGL

EWVSGIGTGGATNYADSVKGRFTISRDNAKNSLYLQMNSLRAEDMA

VYYCARGRYYFDYWGQGTLVTVSS (SEQ ID NO: 48)

SCB-VH-77
EVQLVQSGGGLVHPGGSLRLSCAGSGFTFSRNAMFWVRQAPGEGLE

WVSGIGTGGATNYADSVKGRFTISRDNAKNSLYLQMNSLRAEDMA

VYYCARGRYYFDYWGQGTLVTVSS (SEQ ID NO: 49)

SCB-VH-78
EVQLVQSGGGLVHPGGSLRLSCAGSGFTFSRNAMFWVRQAPGKGLE

WVSGIGTGGATNYADSVKGRFTISRDEAKNSLYLQMNSLRAEDMAV

YYCARGRYYFDYWGQGTLVTVSS (SEQ ID NO: 50)

SCB-VH-79
EVQLVQSGGGLVHPGGSLRLSCAGSGFTFSRNAMFWVRQAPGKGLE

WVSGIGTGGATNYADSVKGRFTISRDNAKNSLYLQMNSLRAGDMA

VYYCARGRYYFDYWGQGTLVTVSS (SEQ ID NO: 51)

SCB-VH-80
EVQLVQSGGGLVHPGGSLRLSCAGSGFTFDDYAMFWVRQAPGKGL

EWVSGIGTGGATNYADSVKGRFTISRDNAKNSLYLQMNSLRAEDMA

VYYCARGRYYFDYWGQGTLVTVSS (SEQ ID NO: 52)

SCB-VH-81
EVQLVQSGGGLVQPGGSLRLSCAASGFTFSRNAMFWVRQAPGKGLE

WVSGIGTGGATSYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV

YYCARGRYYFPWWGQGTLVTVSS (SEQ ID NO: 53)

SCB-VH-82
EVQLVESGGGLVHPGGSLRLSCAASGFTFSRNAMFWVRQAPGKGLE

WVSGIGTGGATSYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV

YYCARGRYYFPWWGQGTLVTVSS (SEQ ID NO: 54)

SCB-VH-83
EVQLVESGGGLVQPGGSLRLSCAGSGFTFSRNAMFWVRQAPGKGLE

WVSGIGTGGATSYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV

YYCARGRYYFPWWGQGTLVTVSS (SEQ ID NO: 55)

SCB-VH-84
EVQLVESGGGLVQPGGSLRLSCAASGFTFSRNAMFWVRQAPGKGLE

WVSGIGTGGATNYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV

YYCARGRYYFPWWGQGTLVTVSS (SEQ ID NO: 56)

SCB-VH-85
EVQLVESGGGLVQPGGSLRLSCAASGFTFSRNAMFWVRQAPGKGLE

WVSGIGTGGATSYADSVKGRFTISRDNAKNSLYLQMNSLRAEDMAV

YYCARGRYYFPWWGQGTLVTVSS (SEQ ID NO: 57)

SCB-VH-86
EVQLVQSGGGLVHPGGSLRLSCAASGFTFSRNAMFWVRQAPGKGLE

WVSGIGTGGATSYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV

YYCARGRYYFPWWGQGTLVTVSS (SEQ ID NO: 58)

SCB-VH-87
EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSRNAMFWVRQAPGKGLE

WVSGIGTGGATSYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV

YYCARGRYYFPWWGQGTLVTVSS (SEQ ID NO: 59)

SCB-VH-88
EVQLVESGGGLVHPGGSLRLSCAGSGFTFSRNAMFWVRQAPGKGLE

WVSGIGTGGATSYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV

YYCARGRYYFPWWGQGTLVTVSS (SEQ ID NO: 60)

SCB-VH-89
EVQLVQSGGGLVHPGGSLRLSCAGSGFTFSRNAMFWVRQAPGKGLE

WVSGIGTGGATSYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV

YYCARGRYYFPWWGQGTLVTVSS (SEQ ID NO: 61)

SCB-VH-90
EVQLVESGGGLVQPGGSLRLSCAASGFTFSRNAMFWVRQAPGKGLE

WVSGIGTGGATNYADSVKGRFTISRDNAKNSLYLQMNSLRAEDMA

VYYCARGRYYFPWWGQGTLVTVSS (SEQ ID NO: 62)

SCB-VH-91
EVQLVESGGGLVQPGGSLRLSCAASGFTFSRNAMFWVRQAPGKGLE

WVSGIGTGGATSYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV

YYCARGRYYFDYWGQGTLVTVSS (SEQ ID NO: 63)

SCB-VH-92
EVQLVQSGGGLVHPGGSLRLSCAGSGFTFSRNAMFWVRQAPGKGLE

WVSGIGTGGATNYADSVKGRFTISRDNAKNSLYLQMNSLRAEDMA

VYYCARGRYYFDYWGQGTLVTVSS (SEQ ID NO: 64)

SCB-VH-93
EVQLVESGGGLVQPGGSLRLSCAASGFTFSRNAMFWVRQAPGKGLE

WVSGIGTGGATSYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV

YYCARGRYYFPWWGQGTLVTVSS (SEQ ID NO: 65)

SCB-92-
RNAMF (SEQ ID NO: 66)

HCDR1

SCB-92-
GIGTGGATSYADSVKG (SEQ ID NO: 67)

HCDR3

SCB-92-
GRYYFDY (SEQ ID NO: 68)

HCDR3

SCB-55-
RASQSVSSSYLA (SEQ ID NO: 69)

LCDR1

SCB-55-
GASSRAT (SEQ ID NO: 70)

LCDR2

SCB-55-
QQYDHSAGWT (SEQ ID NO: 71)

LCDR3

SCB-54-
GASSRAP (SEQ ID NO: 72)

LCDR2

SCB-44-
QQYGSSPPWT (SEQ ID NO: 73)

LCDR3

In certain embodiments, an antibody or antigen-binding fragment thereof comprises a LCVR/HCVR sequence pair of MEDI-37GL-VL/MEDI-37GL-VH.

In certain embodiments, an antibody or antigen-binding fragment thereof comprises an HCVR comprising an HCDR1 sequence of MEDI-37GL-HCDR1, an HCDR2 sequence of MEDI-37GL-HCDR2, and an HCDR3 sequence of MEDI-37GL-HCDR3, and an LCVR comprising an LCDR1 sequence of MEDI-37GL-LCDR1, an LCDR2 sequence of MEDI-37GL-LCDR2, and an LCDR3 sequence of MEDI-37GL-LCDR3.

The antibodies recited below in Table 2 are described in more detail in U.S. Pat. No. 8,877,189, incorporated herein by reference in its entirety for all purposes.

TABLE 2

Sequence
Sequence ID

MEDI-1-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKWWLDYWGKGTLVTVSS (SEQ ID NO: 74)

MEDI-1-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYYCGTWDT

SLSANYVFGTGTKLTVL (SEQ ID NO: 75)

MEDI-2-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKWWLYNWGKGTLVTVSS (SEQ ID NO: 80)

MEDI-2-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYYCGTWDT

SQPPNPLFGTGTKLTVL (SEQ ID NO: 76)

MEDI-3-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKLLKNPWGKGTLVTVSS (SEQ ID NO: 77)

MEDI-3-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYYCGTWFG

TPASNYVFGTGTKLTVL (SEQ ID NO: 78)

MEDI-4-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKWWLYNWGKGTLVTVSS (SEQ ID NO: 80)

MEDI-4-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYYCGTWDT

SSPPQPIFGTGTKLTVL (SEQ ID NO: 79)

MEDI-5-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKWWLYDWGKGTLVTVSS (SEQ ID NO: 80)

MEDI-5-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYYCGTWDT

SSPPQPIFGTGTKLTVL (SEQ ID NO: 79)

MEDI-6-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKYWMYDWGKGTLVTVSS (SEQ ID NO: 81)

MEDI-6-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYYCGTWDT

STTYHPIFGTGTKLTVL (SEQ ID NO: 82)

MEDI-7-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKWWWQYWGKGTLVTVSS (SEQ ID NO: 83)

MEDI-7-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYYCGTWDT

SSPPQPIFGTGTKLTVL (SEQ ID NO: 79)

MEDI-8-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKWWWQYWGKGTLVTVSS (SEQ ID NO: 83)

MEDI-8-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYYCGTWDT

STTYHPIFGTGTKLTVL (SEQ ID NO: 82)

MEDI-9-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKWWLYNWGKGTLVTVSS (SEQ ID NO: 80)

MEDI-9-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYYCGTWDT

STTMYPLFGTGTKLTVL (SEQ ID NO: 84)

MEDI-10-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKWWLYDWGKGTLVTVSS (SEQ ID NO: 80)

MEDI-10-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYYCGTWDT

STVLTPIFGTGTKLTVL (SEQ ID NO: 85)

MEDI-11-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKWWFYDWGKGTLVTVSS (SEQ ID NO: 86)

MEDI-11-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYYCGTWDT

SPSMIPLFGTGTKLTVL (SEQ ID NO: 87)

MEDI-12-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKWWFYDWGKGTLVTVSS (SEQ ID NO: 86)

MEDI-12-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYYCGTWDT

STTMYPLFGTGTKLTVL (SEQ ID NO: 84)

MEDI-13-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKWWLYDWGKGTLVTVSS (SEQ ID NO: 80)

MEDI-13-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYYCGTWDT

STTLQPLFGTGTKLTVL (SEQ ID NO: 88)

MEDI-14-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKWWLYNWGKGTLVTVSS (SEQ ID NO: 80)

MEDI-14-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYYCGTWDT

SPPTKPLFGTGTKLTVL (SEQ ID NO: 89)

MEDI-15-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKWWLYNWGKGTLVTVSS (SEQ ID NO: 80)

MEDI-15-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYYCGTWDT

STHRHPLFGTGTKLTVL (SEQ ID NO: 90)

MEDI-16-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKWWLYNWGKGTLVTVSS (SEQ ID NO: 80)

MEDI-16-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYYCGTWDT

STTYHPIFGTGTKLTVL (SEQ ID NO: 82)

MEDI-17-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKWWWQHWGKGTLVTVSS (SEQ ID NO: 91)

MEDI-17-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYYCGTWDT

SPVDRPIFGTGTKLTVL (SEQ ID NO: 92)

MEDI-18-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKWWWQHWGKGTLVTVSS (SEQ ID NO: 91)

MEDI-18-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYYCGTWDT

STTPMPVFGTGTKLTVL (SEQ ID NO: 93)

MEDI-19-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKWWWQHWGKGTLVTVSS (SEQ ID NO: 91)

MEDI-19-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYYCGTWDT

STTYHPIFGTGTKLTVL (SEQ ID NO: 82)

MEDI-20-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKYWMYDWGKGTLVTVSS (SEQ ID NO: 81)

MEDI-20-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYYCGTWDT

STVWEWPFGTGTKLTVL (SEQ ID NO: 94)

MEDI-21-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSASYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKYWMYDWGKGTLVTVSS (SEQ ID NO: 99)

MEDI-21-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEAVYFCGTWDT

STVWEWPFGTGTKLTVL (SEQ ID NO: 95)

MEDI-22-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKYWMYDWGKGTLVTVSS (SEQ ID NO: 81)

MEDI-22-VL
QPVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYFCGTWDT

STVWEWPFGTGTKLTVL (SEQ ID NO: 96)

MEDI-23-VH
QVQLVQSGAEVRKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKYWMYDWGKGTLVTVSS (SEQ ID NO: 97)

MEDI-23-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSNIGNNYVSWYQQLPGTAPKL

LIYDNNKRPPGIPDRFSGSKSGTSATLAITGLQTGDEADYYCGTWDT

STVWEWPFGTGTKLTVL (SEQ ID NO: 98)

MEDI-24-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPRGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKYWMYDWGKGTLVTVSS (SEQ ID NO: 99)

MEDI-24-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYFCGTWDT

STVWEWPFGTGTKLTVL (SEQ ID NO: 100)

MEDI-25-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPRGGSASYAQKFQGRVSMTRDTSTSTVYMELSSLRSED

TAVYYCARGKYWMYDWGKGTLVTVSS (SEQ ID NO: 101)

MEDI-25-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTTATLAITGLQTGDEADYYCGTWVT

STVWEWPFGTGTKLTVL (SEQ ID NO: 102)

MEDI-26-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKYWMYDWGKGTLVTVSS (SEQ ID NO: 81)

MEDI-26-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYFCGTWDT

STVWEWPFGTGTKLTVL (SEQ ID NO: 100)

MEDI-27-VH
QVQLVQSGAEVRKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRPED

TAVYYCARGKYWMYDWGKGTQVTVSS (SEQ ID NO: 103)

MEDI-27-VL
QSVLTQPPLVSAAPGQKVTISCSGGSSNIGNSYVSWYQRLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYYCGTWDT

STVWEWPFGTGTKLTVL (SEQ ID NO: 104)

MEDI-28-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKYWMYDWGNGTLVTVSS (SEQ ID NO: 105)

MEDI-28-VL
LPVLTQPPSVSAAPGQKVTISCSGGSSSIGNSYVSWYQQLPGAAPKL

LIYDNNKRPSGIPDRFSGFRSGTSATLAITGLQTGDEADYYCGTWDT

SPVWEWPFGTGTKLTVL (SEQ ID NO: 106)

MEDI-29-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKYWMYDWGKGTRVTVSS (SEQ ID NO: 107)

MEDI-29-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYYCGTWDT

SPVWEWPFGTGTKLTVL (SEQ ID NO: 108)

MEDI-30-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKYWMYDWGKGTLVTVSS (SEQ ID NO: 81)

MEDI-30-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQRLPGAAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYYCGTWDT

STVWEWPFGTGTKLTVL (SEQ ID NO: 109)

MEDI-31-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKYWMYDWGKGTLVTVSS (SEQ ID NO: 81)

MEDI-31-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSSIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYYCGTWAT

SPVWEWPFGTGTKLTVL (SEQ ID NO: 110)

MEDI-32-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKYWMYDWGKGTLVTVSS (SEQ ID NO: 81)

MEDI-32-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYFCGTWDT

STAWEWPFGTGTKLTVL (SEQ ID NO: 111)

MEDI-33-VH
QVQLVQSGAEEKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKYWMYDWGKGTLVTVSS (SEQ ID NO: 112)

MEDI-33-VL
QSALTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYFCGTWDT

STVWEWPFGTGTKLTVL (SEQ ID NO: 113)

MEDI-34-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSTSYAQKFQGRVSMTRDTSTSTVYMELSSLRSEDT

AVYYCARGKYWMYDWGKGTLVTVSS (SEQ ID NO: 114)

MEDI-34-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYFCGTWDT

STVWEWPFGTGTKLTVL (SEQ ID NO: 100)

MEDI-35-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKYWMYDWGKGTLVTVSS (SEQ ID NO: 81)

MEDI-35-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYYCGTWDT

SPVWEWPFGTGTKLTVL (SEQ ID NO: 108)

MEDI-36-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSASYAQKFQGR VTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKYWMYDWGKGTLVTVSS (SEQ ID NO: 99)

MEDI-36-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYYCGTWDS

STVWEWPFGTGTKLTVL (SEQ ID NO: 100)

MEDI-37-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPRGGSTSYAQKFQGRVAMTRDTSTSTVYMELSSLRPED

TAVYYCARGKYWMYDWGKGTLVTVSS (SEQ ID NO: 115)

MEDI-37-VL
QSVLTQPPSVSAAPGQKVTISCSGGGSSIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGVPDRFSGSKSGTSATLAITGLQTGDEADYYCGTWD

TSPVWEWPFGTGTKLTVL (SEQ ID NO: 116)

MEDI-38-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSASYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKYWMYDWGKGTLVTVSS (SEQ ID NO: 99)

MEDI-38-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYFCGTWDT

STVWEWPFGTGTKLTVL (SEQ ID NO: 100)

MEDI-39-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPRGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKYWMYDWGKGTLVTVSS (SEQ ID NO: 99)

MEDI-39-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYYCGTWDT

STAWEWPFGTGTKLTVL (SEQ ID NO: 117)

MEDI-40-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKYWMYDWGKGTLVTVSS (SEQ ID NO: 81)

MEDI-40-VL
QSVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQQLPGTAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYYCGTWDS

STVWEWPFGTGTKLTVL (SEQ ID NO: 100)

MEDI-41-VH
QVQLVQSGAEVRKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRPED

TAVYYCARGKYWMYDWGKGTLVTVSG (SEQ ID NO: 118)

MEDI-41-VL
QSVLTQPPSVSAAPGQKVTISCSGGSTNIGNSYVSWYQRLPGTAPKL

LIYDNNKRPPGIPDRFSGSKSGTSATLAITGLQTGDEADYYCGTWDT

STVWEWPFGTGTKLTVL (SEQ ID NO: 119)

MEDI-42-VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWARQAPGQG

LEWVGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSGDT

AVYYCARGKYWMYDWGKGTLVTVSS (SEQ ID NO: 120)

MEDI-42-VL
QAVLTQPPSVSAAPGQKVTISCSGGSSNIGNSYVSWYQRLPGAAPKL

LIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYYCGTWDT

STGWEWPFGTGTKLTVL (SEQ ID NO: 121)

MEDI-37GL-
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWVRQAPGQG

VH
LEWMGIINPRGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED

TAVYYCARGKYWMYDWGKGTLVTVSS (SEQ ID NO: 122)

MEDI-37GL-
QSVLTQPPSVSAAPGQKVTISCSGGGSSIGNSYVSWYQQLPGTAPKL

VL
LIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDT

SPVWEWPFGTGTKLTVL (SEQ ID NO: 123)

MEDI-37GL-
SYYMH (SEQ ID NO: 124)

HCDR1

MEDI-37GL-
IINPRGGSTSYAQKFQG (SEQ ID NO: 125)

HCDR2

MEDI-37GL-
GKYWMYD (SEQ ID NO: 126)

HCDR3

MEDI-37GL-
SGGGSSIGNSYVS (SEQ ID NO: 127)

LCDR1

MEDI-37GL-
DNNKRPS (SEQ ID NO: 128)

LCDR2

MEDI-37GL-
GTWDTSPVWEWP (SEQ ID NO: 129)

LCDR3

In certain embodiments, an antibody or antigen-binding fragment thereof comprises a LCVR/HCVR sequence pair of AJOU-90-VL/AJOU-83-VH.

In certain embodiments, an antibody or antigen-binding fragment thereof comprises an HCVR comprising an HCDR1 sequence of AJOU-84-HCDR1, an HCDR2 sequence of AJOU-85-HCDR2, and an HCDR3 sequence of AJOU-32-HCDR3, and an LCVR comprising an LCDR1 sequence of AJOU-96-LCDR1, an LCDR2 sequence of AJOU-60-LCDR2, and an LCDR3 sequence of AJOU-68-LCDR3.

The antibodies recited below in Table 3 are described in more detail in WO2020/096381 and Kim et al. (Scientific Reports. 9: 7772. 2019), incorporated herein by reference in their entireties for all purposes.

TABLE 3

Sequence ID
Sequence

AJOU-1-VH
EVQLLESGGGLVQPGGSLRLSCAVSGFTFSNYAMSWVRQAPGKGL

EWVSAISSGGGNIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT

AVYYCAKLRRYFDYWGQGTLVTVSS (SEQ ID NO: 130)

AJOU-2-VH
EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKGL

EWVSAISSGGSSIYYADSVKGRFTISRDNSKNTLHLQMNSLRAEDT

AVYYCARGPQRSATAVFDYWGQGTLVTVSS (SEQ ID NO: 131)

AJOU-3-VH
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGL

EWVSWISPNSGNIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT

AVYYCARRPLSAAWSHSSYYNAMDVWGQGTLVTVSS (SEQ ID

NO: 132)

AJOU-4-VH
EVQLLESGGGLVQPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGL

EWVSLISHSGSNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT

AVYYCARPHRAFDYWGQGTLVTVSS (SEQ ID NO: 133)

AJOU-5-VH
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGL

EWVSGISHGSGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT

AVYYCARPHRAFDYWGQGTLVTVSS (SEQ ID NO: 134)

AJOU-6-VH
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGL

EWVSGISHGNGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT

AVYYCAKTGRHFDYWGQGTLVTVSS (SEQ ID NO: 135)

AJOU-7-VH
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGL

EWVSSISPSGSSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA

VYYCARSYRAFDYWGQGTLVTVSS (SEQ ID NO: 136)

AJOU-8-VH
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGL

EWVSAISPSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT

AVYYCARAKRAFDYWGQGTLVTVSS (SEQ ID NO: 137)

AJOU-9-VH
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGL

EWVSAISPGSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT

AVYYCAKFRRHFDYWGQGTLVTVSS (SEQ ID NO: 138)

AJOU-10-VH
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGL

EWVSAISSGGGNIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT

AVYYCARVHRAFDYWGQGTLVTVSS (SEQ ID NO: 139)

AJOU-69-VH
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGL

EWVSAITSSGRSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT

AVYYCARVHRAFDYWGQGTLVTVSS (SEQ ID NO: 140)

AJOU-70-VH
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGL

EWVSAITSSGANIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT

AVYYCARVHRAFDYWGQGTLVTVSS (SEQ ID NO: 141)

AJOU-71-VH
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGL

EWVSAITSSGGNIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT

AVYYCARVHRAFDYWGQGTLVTVSS (SEQ ID NO: 142)

AJOU-72-VH
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGL

EWVSAITAGGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT

AVYYCARVHRAFDYWGQGTLVTVSS (SEQ ID NO: 143)

AJOU-83-VH
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRHAMAWVRQAPGKGL

EWVSAITSSGRSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT

AVYYCARVHRAFDYWGQGTLVTVSS (SEQ ID NO: 144)

AJOU-33-VL
QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVNWYQQLPGTAPK

LLIYDNSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGTW

DASLSAYVFGGGTKLTVL (SEQ ID NO: 145)

AJOU-34-VL
QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNNVSWYQQLPGTAPKL

LIYANSKRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGSWD

DSLSAYVFGGGTKLTVL (SEQ ID NO: 146)

AJOU-35-VL
QSVLTQPPSAPGTPGQRVTISCTGSSSNIGSNSVNWYQQLPGTAPKL

LIYDDSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCDAWD

SSLSAYVFGGGTKLTVL (SEQ ID NO: 147)

AJOU-36-VL
QSVLTQPPSASGTPGQRVTLSCTGSSSNIGSNYVSWYQQLPGTAPK

LLIYADSQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGTW

DDSLSGYVFGGGTKLTVL (SEQ ID NO: 148)

AJOU-37-VL
QSVLTQPPSASGTPGQRVTISCSSSSSNIGSNYVSWYQQLPGTAPKL

LIYSDSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGSWDY

SLSAYVFGGGTKLTVL (SEQ ID NO: 149)

AJOU-38-VL
QSVLTQPPSASGTPGQRVTISCTGSSSNIGNNTVSWYQQLPGTAPKL

LIYDNSHRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCGSWD

YSLSAYVFGGGTKLTVL (SEQ ID NO: 150)

AJOU-39-VL
QSVLTQPPSASGTPGQRVTISCTGSSSNIGNNDVNWYQQLPGTAPK

LLIYYDSQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATW

DASLSAYVFGGGTKLTVL (SEQ ID NO: 151)

AJOU-40-VL
QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNAVNWYQQLPGTAPKL

LIYYDNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGTWD

DSLNGYVFGGGTKLTVL (SEQ ID NO: 152)

AJOU-41-VL
QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNAVTWYQQLPGTAPK

LLIYDDSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGSW

DYSLSAYVFGGGTKLTVL (SEQ ID NO: 153)

AJOU-42-VL
QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTFNWYQQLPGTAPKL

LIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGTWD

YSLSGYVLGGGTKLTVL (SEQ ID NO: 154)

AJOU-77-VL
QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTENWYQQLPGTAPKL

LIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGTWD

YSLSGYVLGGGTKLTVL (SEQ ID NO: 154)

AJOU-78-VL
QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTFNWYQQLPGTAPKL

LIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGTWD

YSLRGYVLGGGTKLTVL (SEQ ID NO: 155)

AJOU-79-VL
QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTFNWYQQLPGTAPKL

LIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGYWD

YSLSGYVLGGGTKLTVL (SEQ ID NO: 156)

AJOU-80-VL
QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTFNWYQQLPGTAPKL

LIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGTWD

YSLSGYVLGGGTKLTVL (SEQ ID NO: 154)

AJOU-86-VL
QSVLTQPPSASGTPGQRVTISCSGSSANSRTDGFNWYQQLPGTAPK

LLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGTW

DYSLSGYVLGGGTKLTVLG (SEQ ID NO: 157)

AJOU-87-VL
QSVLTQPPSASGTPGQRVTISCSGSAQFGSRDNFNWYQQLPGTAPK

LLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGTW

DYSLSGYVLGGGTKLTVLG (SEQ ID NO: 158)

AJOU-88-VL
QSVLTQPPSASGTPGQRVTISCSGSTKQMHNYQFNWYQQLPGTAP

KLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGT

WDYSLSGYVLGGGTKLTVLG (SEQ ID NO: 159)

AJOU-89-VL
QSVLTQPPSASGTPGQRVTISCSGSLLRGENLQFNWYQQLPGTAPK

LLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGTW

DYSLSGYVLGGGTKLTVLG (SEQ ID NO: 160)

AJOU-90-VL
QSVLTQPPSASGTPGQRVTISCSGSPLFPDSGSFNWYQQLPGTAPKL

LIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGTWD

YSLSGYVLGGGTKLTVLG (SEQ ID NO: 161)

AJOU-91-VL
QSVLTQPPSASGTPGQRVTISCSGSAALDLSPSFNWYQQLPGTAPKL

LIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGTWD

YSLSGYVLGGGTKLTVLG (SEQ ID NO: 162)

AJOU-84-
RHAMA (SEQ ID NO: 163)

HCDR1

AJOU-85-
AITSSGRSIYYADSVKG (SEQ ID NO: 164)

HCDR2

AJOU-32-
VHRAFDY (SEQ ID NO: 165)

HCDR3

AJOU-96-
SGSPLFPDSGSFN (SEQ ID NO: 166)

LCDR1

AJOU-60-
ADSHRPS (SEQ ID NO: 167)

LCDR2

AJOU-68-
GTWDYSLSGYV (SEQ ID NO: 168)

LCDR3

In certain embodiments, an antibody or antigen-binding fragment thereof of the disclosure comprises light chain variable region (LCVR) and heavy chain variable region (HCVR) sequence pairs (LCVR/HCVR) selected from the group of sequences listed in Table 4 consisting of 11/3, 27/19, 43/35, 59/51, 75/67, 91/83, 107/99, 123/115, 155/147, and 171/163.

The antibodies recited below in Table 4 are described in more detail in U.S. Pat. Nos. 7,605,237 and 7,608,693, incorporated herein by reference in their entireties for all purposes.

TABLE 4

Sequence ID
Sequence

REGN-VH-3
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGISWVRQAPGQG

LEWMGWISVYNGKTNYAQKLQGRVTMTTDTSTTTAYMEMRSLR

SDDTAVYYCARGSGYDLDYWGQGTLVSVSS (SEQ ID NO: 169)

REGN-VH-19
EVOLVESGGGLVQPGGSLRLSCAASGFTFSSFWMTWVRQAPGKG

LEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRA

EDTAVYYCARDPGRTMVRGGIRYYYGMDVWGQGTTVTVSS

(SEQ ID NO: 170)

REGN-VH-35
EVKLAESGGGLVQPGGSLRLSCAASGFTFSSHWMNWVRQAPGKG

LEWVANIKQDGSDKYYVDSVKGRFTISRDNAKNSLYLQLNSLIAE

DTAVYYCARDRGVRPPRGAFDIWGQGTMVTVSS (SEQ ID NO:

171)

REGN-VH-51
QVQLVQSGAEVKKPGASVKVSCKASGYTFNSYGISWVRQAPGQG

LEWMGWIRTYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLR

SDDTAVYYCARDEARIVVAGTTPYYYGMDVWGQGTTVTVSS

(SEQ ID NO: 172)

REGN-VH-67
QVQLVESGGGLVQPGGSLRLSCAVSGFTISDHYMSWIRQAPGKGL

EWISYISSSGSKIYYADSVKGRFTISRDNAKNSLFLQMNSLRAEDT

AVYYCARTRQLVGDYWGQGTLVTVSS (SEQ ID NO: 173)

REGN-VH-83
EVQLVESGGGLVQPGRSLRLSCAASGFTFDNYAMHWVRQAPGK

GLEWVSGIRWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRA

EDTALYYCAKEGGYSGYRPGPFFDYWGQGTLVTVSS (SEQ ID

NO: 174)

REGN-VH-99
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGISWVRQAPGQG

LEWMGWISVYNGHTNYAQKLQGRVTMTTDTSTSTAYMELRSLR

SDDTAVYYCARGSGYDFDSWGQGTLVTVSS (SEQ ID NO: 175)

REGN-VH-115
QVQLVQSGAEVKKPGASVKVSCKASRYTFTSYDINWVRQATGQG

LEWMGWMNPNSGNTGYAQKFQGRVTMTRNTSTSTAYMELSSLR

SEDTAVYYCARVRRFFDYWGQGTLVTVSS (SEQ ID NO: 176)

REGN-VH-147
QVQLVQSGPEVKKPGASVKVSCKASGYTFTNYGISWVRQAPGQG

LEWMGWISVYNGNINYAQKLQGRVTMTTDTSTSTAYMDLRSLRS

DDTAVYYCARGSGYDFDYWGQGTLVTVSS (SEQ ID NO: 177)

REGN-VH-163
QVQLVQSGAEVKKPGASVKVSCKDSAYTFNRYGISWVRQAPGQG

LEWMGWISAYTGNTVYAQKLQGRVTMTTDNSTSTAYMELRSLR

SDDTAVYYCARDKSIFGVVRGFDYWGQGTLVTVSS (SEQ ID NO:

178)

REGN-VL-11
AIQMTQSPSSLSASVGDRVTITCRASQGIRNALGWYQQKPGKAPK

LLIYAASSLQSGVPSRFSGSGSGTDFTLTFSSLQPEDFATYYCLQDF

NYPYTFGQGTKLEIK (SEQ ID NO: 179)

REGN-VL-27
DIQMTQSPSSVSASVGDRVTISCRASQGVSSWLAWYQQKPGNAP

KLLISAASSIQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQA

NSFPLTFGGGTKVEIK (SEQ ID NO: 180)

REGN-VL-43
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPK

LLIYAASSFQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQAN

SFPLTFGGGTTVEIK (SEQ ID NO: 181)

REGN-VL-59
DIQMTQSPSSVSASVGDRVTITCRASQDISIWLAWYQQSPGKAPKL

LINVASRLQSGVPSRFSGSGSGTDFTLTINSLQPEDFVTYYCQQAN

SFPITFGQGTRLATK (SEQ ID NO: 182)

REGN-VL-75
DIQLTQSPSFLSASVGDRVTITCWASQGISSYLAWYQQKPGKAPKL

LIFAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNS

YPLTFGGGTKVEIR (SEQ ID NO: 183)

REGN-VL-91
EIVMTQSPATLSVSPGERATLSCRASQSVNYNLAWYQHKPGQAPR

LLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYN

NWPLTFGGGTKVEIK (SEQ ID NO: 184)

REGN-VL-107
AIQMTQSSSSLSASVGDRVTITCRASQAIRNALGWYQQKPGKAPK

VLIYAASSLQSGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDY

DYPYTFGQGTKLEIK (SEQ ID NO: 185)

REGN-VL-123
DIQLTQSPSFLSASVGDRVTITCWASQGIISYLAWYQQKPGKAPKL

LIYAASTLHSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCHQLKS

YPITFGQGTRLEIK (SEQ ID NO: 186)

REGN-VL-155
AIQMTQSPSSLSASVGDRVTITCRASQDIRNALGWYQQKPGKAPK

LLIYAASSLQSGVPSRFSGSASGTDFTLTISSLQPEDFAAYYCLQDY

NYPYTFGQGTKLEIK (SEQ ID NO: 187)

REGN-VL-171
EIVMTQSPVTLSLSPGERATLPCRASQSVSSSLAWYQQKAGQSPRL

LIYGASTRATGIPARFSGSGSGTEFTLTISNLQSEDFAVYYCQQYN

NWPLTFGGGTKVEIK (SEQ ID NO: 188)

It is understood that the present disclosure is not limited to any of the aforesaid protein(s), protein(s) of interest, antibody(s), cell culture media, host cell protein(s), protein alkylating agent(s), protein denaturing agent(s), protein reducing agent(s), digestive enzyme(s), treatment(s), process(es), sample(s), surfactant(s), detergent(s), chromatographic method(s), filtration method(s), mass spectrometer(s), database(s), bioinformatics tool(s), pH, temperature(s), or concentration(s), and any protein(s), protein(s) of interest, antibody(s), cell culture media, host cell protein(s), protein alkylating agent(s), protein denaturing agent(s), protein reducing agent(s), digestive enzyme(s), treatment(s), process(es), sample(s), surfactant(s), detergent(s), chromatographic method(s), filtration method(s), mass spectrometer(s), database(s), bioinformatics tool(s), pH, temperature(s), or concentration(s) can be selected by any suitable means.

The present disclosure will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention.

Cell Culture Media and Additives

The elimination of serum and reduction or elimination of hydrolysates from cell culture media, while reducing lot-to-lot variability and enhancing downstream processing steps, unfortunately diminishes cell growth, viability and protein expression. Thus, chemically defined serum-free and low-to-no-hydrolysate media requires additional ingredients to improve cell growth and protein production. The cell culture media of the invention may be supplemented with additional ingredients such as polyamines or increased concentrations of components like amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, trace elements and the like, depending on the requirements of the cells to be cultured or the desired cell culture parameters.

In some embodiments, the cell culture medium disclosed herein is supplemented with polyamines (“PS media”) such as ornithine, putrescine, spermine, spermidine, or combinations to improve cell growth, cell viability, and recombinant protein production.

In one aspect, the PS media comprises ornithine at a concentration (expressed in micromoles per liter) of at least about 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 540, 545, 550, 555, 560, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 625, 630, 635, 640, 645, 650, 700, 750, 800, 850, or 900±14 μM. In one aspect, the PS media comprises ornithine at a concentration between about 0.09 and 0.9 mM.

In one embodiment and in addition to the inclusion of ornithine or putrescine, the media comprises a mixture of nucleosides in a cumulative concentration of at least 50 μM, at least 60 μM, at least 70 μM, at least 80 μM, at least 90 μM, at least 100 μM, at least 110 μM, at least 115 μM, at least 120 μM, at least 125 μM, at least 130 μM, at least 135 μM, at least 140 μM, at least 145 μM, at least 150 μM, at least 155 μM, at least 160 μM, at least 165 μM, or at least 170 μM.

In one embodiment, the media comprises about 174 μM±26 μM nucleoside. In one embodiment, the media comprises purine derivatives in a cumulative concentration of at least 40 μM, at least 45 μM, at least 50 μM, at least 55 μM, at least 60 μM, at least 65 μM, at least 70 μM, at least 75 μM, at least 80 μM, at least 85 μM, at least 90 μM, at least 95 μM, at least 100 μM, or at least 105 μM. In one embodiment, the media comprises about 106 μM±5 μM of purine derivatives. Purine derivatives include hypoxanthine and the nucleosides adenosine and guanosine. In one embodiment, the media comprises pyrimidine derivatives in a cumulative concentration of at least 30 μM, at least 35 μM, at least 40 μM, at least 45 μM, at least 50 μM, at least 55 μM, at least 60 μM, or at least 65 μM. In one embodiment, the media comprises about 68 μM±5 μM of pyrimidine derivatives. Pyrimidine derivatives include the nucleosides thymidine, uridine, and cytidine. In one particular embodiment, the media comprises adenosine, guanosine, cytidine, uridine, thymidine and hypoxanthine.

In addition to the inclusion of ornithine or putrescine, in one embodiment, the media also comprises amino acids in a cumulative concentration of at least 40 mM, wherein the amount of glutamine is not included in the calculation of the cumulative total. In one embodiment, glutamine is not included in the media, but may be supplied as a “point-of-use addition” to the media during the culturing of cells, such as during the production of protein. Thus, in some embodiments, such as in the method to culture cells or the method to produce a protein of interest, the media may be supplemented with glutamine as a point-of-use addition. In one such embodiment, glutamine is added in an amount less than about 40 mM, less than about 35 mM, less than about 30 mM, less than about 25 mM, less than about 20 mM, less than about 15 mM, less than about 10 mM, less than about 8 mM, less than about 7 mM, less than about 6 mM, less than about 5 mM, less than about 4 mM, less than about 3 mM, or less than about 2.5 mM. In one embodiment, the amount of glutamine in the media that was supplemented with glutamine is about 2 mM±0.5 mM.

In one embodiment, in addition to the inclusion of ornithine or a combination of both ornithine and putrescine, the media also comprises amino acids having a non-polar side group in a concentration of at least 15 mM, at least 24 mM, at least 25 mM, at least 26 mM, at least 27 mM, at least 28 mM, at least 29 mM, or at least 30 mM. In one embodiment, the media comprises about 30 mM of amino acids having a non-polar side group. In one embodiment, of the total amount of amino acids by mole contained within the media, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, or at least 41% are amino acids having non-polar side groups. In one embodiment, about 42% 1% by mole of the amino acids in the media are amino acids having a non-polar side group. Amino acids having a non-polar side group include alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, and methionine. In one embodiment, the optional mixture of amino acid supplements are selected from the group consisting of the amino acids in Table 5 below:

TABLE 5

Amino acid
RANGE mM (mmol/L)
RANGE (g/L)

Alanine
0-11.2
0-1

Arginine
2.4-11.9
0.5-2.5

Asparagine
1.3-33.3
0.2-5

Aspartic Acid
1.5-93.9
0.2-12.5

Cysteine
1.1-19.9
0.2-3.5

Glutamic acid
1.4-47.6
0.2-7

Glutamine
0-23.9
0-3.5

Glycine
0-16.7
0-1.25

Histidine
1-9.5
0.2-2

Isoleucine
1.5-22.9
0.2-3

Leucine
1.5-38.1
0.2-5

Lysine
2.7-24.6
0.5-4.5

Methionine
1.3-13.4
0.2-2

Phenylalanine
1.2-18.2
0.2-3

Proline
1.7-26.1
0.2-3

Serine
1.9-57.1
0.2-6

Threonine
1.7-33.6
0.2-4

Tryptophan
0.5-14.7
0.1-3

Tyrosine
0.9-22.2
0.2-5

Valine
1.7-34.1
0.2-4

In one embodiment, in addition to the inclusion of ornithine or a combination of both ornithine and putrescine, the media also comprises amino acids having an uncharged polar side group in a concentration of about 10 mM to 34 mM, about 11 mM to 33 mM, about 12 mM to 32 mM, about 13 mM to 31 mM, about 14 mM to 30 mM, about 15 mM to 29 mM, about 16 mM to 28 mM, about 17 mM to 27 mM, about 18 mM to 26 mM, about 19 mM to 25 mM, about 20 mM to 24 mM, about 21 mM to 23 mM, about 22 mM, about 23 mM, about 24 mM, or about 25 mM. In one embodiment, the medium comprises about 22 mM of amino acids having an uncharged polar side group. In another embodiment, the medium comprises about 12 mM amino acids having an uncharged polar side group. In one embodiment, of the total amount by mole of amino acids contained within the media, about 14% to 46%, about 15% to 45%, about 16% to 44%, about 17% to 043%, about 18% to 42%, about 19% to 41%, about 20% to 40%, about 21% to 39%, about 22% to 38%, about 23% to 37%, about 24% to 36%, about 25% to 35%, about 26% to 34%, about 27% to 33%, about 28% to 32%, about 29% to 31%, or about 30% are amino acids having uncharged polar side groups. In one embodiment, about 30%±30% by mole of the amino acids in the media are amino acids having an uncharged polar side group. Amino acids having an uncharged polar side group include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.

In one embodiment, in addition to the inclusion of ornithine or a combination of both ornithine and putrescine, the media also comprises amino acids having a negative charge at pH 6 (e.g., acidic amino acids) in a concentration of about 4 mM to 14 mM, about 5 mM to 13 mM, about 6 mM to 12 mM, about 7 mM to 11 mM, about 8 mM to 10 mM, about 9 mM, or about 4 mM. In one embodiment, the media comprises about 9 mM of acidic amino acids. In one embodiment, the media comprises 9 mM±1 mM of acidic amino acids. In one embodiment, of the total amount by mole of amino acids contained within the media, about 8% to 18%, about 9% to 17%, about 10% to 16%, about 11% to 15%, about 12% to 14%, or about 13% are acidic amino acids. In one embodiment, about 12.6%±1% by mole of the amino acids in the media are acidic amino acids. Acidic amino acids include aspartic acid and glutamic acid.

In one embodiment, in addition to the inclusion of ornithine or a combination of both ornithine and putrescine, the media also comprises amino acids having a positive charge at pH 6 (e.g., basic amino acids) in a concentration of at least 3.5 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM, at least 10 mM, or at least 11 mM. In one embodiment, the media comprises about 11 mM of basic amino acids. In one embodiment, the media comprises about 11.42 mM±1 mM of basic amino acids. In one embodiment, of the total amount by mole of amino acids contained within the media, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, or at least 15% are basic amino acids. In one embodiment, about 16% by mole of the amino acids in the media are basic amino acids. In one embodiment, about 15.8% 2.4% by mole of the amino acids in the media are basic amino acids. In one embodiment, about 21%±3.2% by mole of the amino acids in the media are basic amino acids. Basic amino acids include lysine, arginine, and histidine.

In one embodiment, in addition to the inclusion of ornithine or a combination of both ornithine and putrescine, the media also comprises about 30 mM non-polar amino acids, about 22 mM uncharged polar amino acids, about 9 mM acidic amino acids, and about 11 mM basic amino acids. In one embodiment, of the amino acids in the media, about 42% by mole are non-polar amino acids, about 30% by mole are uncharged polar amino acids, about 13% by mole are acidic amino acids, and about 16% by mole are basic amino acids.

In addition to the inclusion of ornithine or a combination of both ornithine and putrescine, in one embodiment, the media comprises micromolar amounts of fatty acids (or fatty acid derivatives) and tocopherol. In one embodiment, the fatty acids include any one or more of linoleic acid, linolenic acid, thioctic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, arachidonic acid, lauric acid, behenic acid, decanoic acid, dodecanoic acid, hexanoic acid, lignoceric acid, myristic acid, and octanoic acid. In one embodiment, the media comprises tocopherol, linoleic acid, and thioctic acid.

In one embodiment, the media also comprises a mixture of vitamins, which includes other nutrients and essential nutrients, at a cumulative concentration of at least about 700 μM or at least about 2 mM. In one embodiment, the mixture of vitamins comprises one or more of D-biotin, choline chloride, folic acid, myo-inositol, niacinamide, pyridoxine HCl, D-pantothenic acid (hemiCa), riboflavin, thiamine HCl, vitamin B12, and the like. In one embodiment, the mixture of vitamins includes all of D-biotin, choline chloride, folic acid, myo-inositol, niacinamide, pyridoxine HCl, D-pantothenic acid (hemiCa), riboflavin, thiamine HCl, and vitamin B12.

In one embodiment, the media also comprises taurine, hypotaurine, or combinations thereof. Taurine is present in many tissues of humans and other mammalian species, e.g. brain, retina, myocardium, skeletal and smooth muscle, platelets and neutrophils. Taurine may help with osmoregulation, membrane stabilization and anti-inflammation, and also regulates mitochondrial protein synthesis through enhanced electron transport chain activity that protects against superoxide generation (Jong et al., 2010, Journal of Biomedical Science 17(Suppl 1):S25; Jong et al., 2012, Amino Acids 42:2223-2232sw). In one aspect, a cell culture medium which is serum-free may comprise about 0.1 mM to about 10 mM taurine, about 0.1 mM to about 1 mM taurine, about 0.4 mM to about 8 mM taurine, about 1 mM to about 6 mM taurine, about 2 mM to about 5 mM taurine, or about 3 to about 4 mM taurine. Taurine has also been found to increase cellular specific productivity and allows for lower ammonia byproduct by those cells to improve the production of recombinant proteins. See U.S. Pat. No. 10,927,342, which is incorporated by reference in its entirety.

In one embodiment, said Dupilumab is produced at a titer of at least 1.5 g/L in said cell culture production medium by day 4. In another embodiment, said Dupilumab is produced at a titer of at least 2 g/L in said cell culture production medium by day 4.

In one embodiment, said culturing lasts for about 10-18 days. In another embodiment, said culturing lasts for about 10 days, about 12 days, about 14 days, about 16 days, or about 18 days.

In one embodiment, said cell culture production medium is a serum-free medium. In some embodiments, said serum-free medium comprises a recombinant growth factor, an osmolarity regulator, a pH buffer agent, glutamine, and a cell protectant.