(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(19) World Intellectual Property
`Organization
`International Bureau
`
`(43) International Publication Date
`10 March 2016 (10.03.2016)
`
`WIPOI PCT
`
`\9
`
`(10) International Publication Number
`
`WO 2016/036916 A1
`
`(51)
`
`International Patent Classification:
`C12P 21/00 (2006.01)
`C12N 5/0783 (2010.01)
`C12P 21/08 (2006.01)
`C07K 16/28 (2006.01)
`C121V15/80 (2006.01)
`C07K 16/46 (2006.01)
`C12N 15/85 (2006.01)
`
`(21)
`
`International Application Number:
`
`PCT/US2015/048258
`
`(22)
`
`International Filing Date:
`
`(25)
`
`(26)
`
`(30)
`
`(71)
`
`(72)
`
`(74)
`
`(81)
`
`Filing Language:
`
`Publication Language:
`
`3 September 2015 (03.09.2015)
`English
`
`English
`
`Priority Data:
`62/045,207
`
`3 September 2014 (03.09.2014)
`
`US
`
`Applicant: BIOATLA, LLC [US/US]; 1101 1 Torreyana
`Road, San Diego, CA 92121 (US).
`
`Inventor: SHORT, Jay M.; 12985 Via Esperia, Del Mar,
`California 92014 (US).
`
`Agent: DUNLEAVY, Kevin J.; Mendelsohn, Drucker, &
`Dunleavy, P.C., 1500 John F. Kennedy Blvd., Suite 312,
`Philadelphia, Pennsylvania 19102 (US).
`
`Designated States (unless otherwise indicated, for every
`kind ofnational protection available): AE, AG, AL, AM,
`
`A0, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY,
`BZ, CA, CII, CL, CN, CO, CR, CU, CZ, DE, DK, DM,
`DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`IIN, IIR, IIU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR,
`KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG,
`MK, MN, MW, MX, MY, MZ, NA, NG, NI, No, NZ, OM,
`PA, PE, PG, PH, PL, PT, QA, Ro, Rs, RU, RW, SA, SC,
`SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN,
`TR, TT, TZ, UA, UG, Us, UZ, VC, VN, ZA, ZM, ZW.
`
`(84)
`
`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ,
`TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU,
`TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE,
`DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU,
`LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK,
`SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ,
`GW, KM, ML, MR, NE, SN, TD, TG).
`Declarations under Rule 4.17:
`
`ofinventorship (Rule 4.17(iv))
`Published:
`
`with international search report (Art. 21(3))
`
`(54) Title: DISCOVERING AND PRODUCING CONDITIONALLY ACTIVE BIOLOGIC PROTEINS IN THE SAME EUKA-
`RYOTIC CELL PRODUCTION HOSTS
`
`l 51 ASTR
`
`
`
`
`
`
`
`FIG. 1
`
`(57) Abstract: A method of preparing a conditionally act-
`ive biologic protein by selecting a wild-type biologic pro-
`tein, evolving the DNA which encodes the Wild-type bio-
`logic protein using one or more evolutionary techniques
`to create mutant DNAs, expressing the mutant DNAs in a
`eukaryotic cell production host to obtain a mutant protein,
`subjecting the mutant protein and the Wild—type protein to
`an assay under a normal physiological condition and to an
`assay under an aberrant condition, selecting a condition—
`ally active mutant protein which exhibits at least one of:
`(a) a decrease in activity in the assay at
`the normal
`physiological condition compared to the wild-type pro-
`tein, and (b) an increase in activity in the assay under the
`aberrant condition compared to the wild-type protein; and
`producing the conditionally active biologic protein in the
`same eukaryotic cell production host used in the expres-
`sion step.
`
`
`
`W02016/036916A1|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
`
`

`

`WO 2016/036916
`
`PCT/US2015/048258
`
`DISCOVERING AND PRODUCING CONDITIONALLY ACTIVE BIOLOGIC
`
`PROTEINS IN THE SAME EUKARYOTIC CELL PRODUCTION HOSTS
`
`FIELD OF THE DISCLOSURE
`
`[0001] This disclosure relates to the fields of protein evolution and activity. Specifically, this
`
`disclosure relates to a method of generating conditionally active biologic proteins from wild
`
`type proteins, in particular therapeutic proteins, which generated proteins are reversibly or
`
`irreversibly virtually inactivated at normal physiological conditions typically encountered by
`
`the wild—type protein but active at other conditions. For example, evolved proteins are
`
`Virtually inactive at body temperature, but are active at lower temperatures.
`
`BACKGROUND OF THE DISCLOSURE
`
`[0002] There is a considerable body of literature describing the potential for evolving
`
`proteins for a variety of characteristics, especially enzymes for example, to be stabilized for
`
`operation at different conditions. For example, enzymes have been evolved to be stabilized at
`
`higher temperatures, with varying activity. In situations where there is an activity
`
`improvement at the high temperature, a substantial portion of the improvement can be
`
`attributed to the higher kinetic activity commonly described by the Q10 rule where it is
`
`estimated that in the case of an enzyme the turnover doubles for every increase of 10 degrees
`
`Celsius. In addition, there exist examples of natural mutations that destabilize proteins at their
`
`normal operating conditions, such as the activity of the molecule at a given temperature. For
`
`temperature mutants, these mutants can be active at a lower temperature, but typically are
`
`active at a reduced level compared to the wild type molecules from which they are derived.
`
`This may be described by a reduction in activity guided by the Q10 or similar rules.
`
`[0003] It is desirable to generate useful molecules that are conditionally active. For example,
`
`such molecules may be virtually inactive under conditions typically encountered by the
`
`corresponding wild—type molecule from which they are derived, but are active at other
`
`conditions at a level that is equal to or higher than the activity under the conditions typically
`
`encountered by the corresponding wild—type molecule, or that are activated or inactivated in
`
`certain microenvironments, or that are activated or inactivated over time. Besides
`
`temperature, other conditions for which the proteins can be evolved or optimized include pH,
`
`osmotic pressure, osmolality, oxidative stress and electrolyte concentration. Other desirable
`
`properties that can be optimized during evolution include chemical resistance, and proteolytic
`
`resistance.
`
`

`

`WO 2016/036916
`
`PCT/U82015/048258
`
`[0004] Many strategies for evolving or engineering molecules have been published.
`
`However, engineering or evolving a protein to be inactive or virtually inactive (less than 10%
`
`activity and especially 1% activity) at its wild type operating condition, while maintaining
`
`activity equivalent or better than its wild type condition at new conditions, requires that the
`
`destabilizing mutation(s) co—exist with activity increasing mutations that do not counter the
`
`destabilizing effect. It is expected that destabilization would reduce the protein's activity
`
`greater than the effects predicted by standard rules such as Q10, therefore the ability to evolve
`
`proteins that work efficiently at lower temperature, for example, while being inactivated
`
`under their normal operating condition, creates an unexpected new class of proteins we refer
`
`to as Mirac Proteins.
`
`[0005] Throughout this application, various publications are referenced by author and date.
`
`The disclosures of these publications in their entireties are hereby incorporated by reference
`
`into this application in order to more fully describe the state of the art as known to those
`
`skilled therein as of the date of the disclosure described and claimed herein.
`
`SUMMARY OF THE DISCLOSURE
`
`[0006] The disclosure provides a method of preparing a conditionally active biologic protein,
`
`the method comprising the steps of: selecting a wild—type biologic protein, evolving the DNA
`
`which encodes the wild-type biologic protein using one or more evolutionary techniques to
`
`create mutant DNAs, expressing the mutant DNAs in a eukaryotic cell production host to
`
`obtain a mutant protein, subjecting the mutant protein and the wild—type protein to an assay
`
`under a normal physiological condition and to an assay under an aberrant condition, selecting
`
`a conditionally active mutant protein which exhibits at least one of: (a) a decrease in activity
`
`in the assay at the normal physiological condition compared to the wild—type protein, and (b)
`
`an increase in activity in the assay under the aberrant condition compared to the wild—type
`
`protein; and producing the conditionally active biologic protein in the same eukaryotic cell
`
`production host used in the expression step. In another aspect, the conditionally active mutant
`
`protein exhibits both: (a) a decrease in activity in the assay at the normal physiological
`
`condition compared to the wild—type protein, and (b) an increase in activity in the assay under
`
`the aberrant condition compared to the wild—type protein. In various aspects, the normal
`
`physiological condition is selected from one or more of temperature, pH, osmotic pressure,
`
`osmolality, oxidative stress and electrolyte concentration. In a particular aspect, the normal
`
`physiological condition is temperature; wherein the conditionally active biologic protein is
`
`virtually inactive at the normal physiological temperature, but is active at an aberrant
`
`2
`
`

`

`WO 2016/036916
`
`PCT/U82015/048258
`
`temperature less than the normal physiological temperature. In other aspects, the
`
`conditionally active biologic protein is reversibly or irreversibly inactivated at the Wild type
`
`normal physiological conditions. In one specific aspect, the protein is reversibly inactivated at
`
`the wild type normal physiological conditions. Alternatively, conditionally active biologic
`
`proteins are selected from those proteins which exhibit changes in activity, reversibly or
`
`irreversibly, in two or more different physiological conditions.
`
`[0007] In one embodiment, the Wild—type biologic protein is an enzyme, in certain aspects the
`
`wild—type biologic protein is selected from the group consisting of tissue plasminogen
`
`activator, streptokinase, urokinase, renin, and hyaluronidase.
`
`[0008] In another embodiment, the wild—type biologic protein is selected from calcitonin
`
`gene—related peptide (CGRP), substance P (SP), neuropeptide Y (NPY), vasoactive intestinal
`
`peptide (VTP), vasopressin, and angiostatin.
`
`[0009] In another embodiment, the biologic protein is an antibody.
`
`[00010]
`
`In another embodiment, the normal physiological condition in any of the
`
`preceding embodiments is temperature; and the conditionally active biologic protein is
`
`substantially inactive at the normal physiological temperature, and is active at an aberrant
`
`temperature less than the normal physiological temperature.
`
`[00011]
`
`In another embodiment, the evolving step in any of the preceding
`
`embodiments comprises a technique selected from the group consisting of PCR, error—prone
`
`PCR, shuffling, oligonucleotide—directed mutagenesis, assembly PCR, sexual PCR
`
`mutagenesis, in viva mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis,
`
`exponential ensemble mutagenesis, site—specific mutagenesis, gene reassembly, gene site
`
`saturated mutagenesis, in vitro mutagenesis, ligase chain reaction, oligonucleotide synthesis
`
`and combination thereof.
`
`[00012]
`
`In another embodiment, the eukaryotic cell production host in any of the
`
`preceding embodiments is selected from a fungal cell, an insect cell, a mammalian cell, an
`
`adenovirus, and a plant cell.
`
`[00013]
`
`In another embodiment, the mammalian cell in any of the preceding
`
`embodiments is selected from Bowes melanoma cells, COS—7 cells, C127 cells, HeLa cells,
`
`BHK cells, 3T3 mouse fibroblast cells, BHK21 Syrian hamster fibroblast cells, MDCK dog
`
`epithelial cells, PtKl rat kangaroo epithelial cells, SP2/0 mouse plasma cells, NSO mouse
`
`plasma cells, HEK 293 human embryonic kidney cells, COS monkey kidney cells, CHO
`
`cells, CHO—S, R1 mouse embryonic cells, E14.1 mouse embryonic cells, H1 human
`
`embryonic cells, H9 human embryonic cells, and PER C.6, human embryonic cells.
`
`3
`
`

`

`WO 2016/036916
`
`PCT/U82015/048258
`
`[00014]
`
`In another embodiment, the producing step of any of the preceding
`
`embodiments comprises manufacturing.
`
`[00015]
`
`In another embodiment, the conditionally active biologic protein in any of the
`
`preceding embodiments is used as a recognition protein by a viral particle.
`
`[00016]
`
`In another embodiment, the conditionally active biologic protein in any of the
`
`preceding embodiments that binds with a target protein on a target cell is integrated into a
`
`chimeric antigen receptor.
`
`[00017]
`
`In another embodiment, the conditionally active biologic protein prepared by
`
`the method of any of the preceding embodiments is reversibly or irreversibly inactivated at
`
`the normal physiological condition.
`
`[00018]
`
`In another embodiment, the conditionally active biologic protein of the
`
`previous paragraph comprises at least one non—natural amino acid.
`
`[00019]
`
`In another embodiment, the conditionally active biologic protein of the
`
`previous two paragraphs is a part of a chimeric antigen receptor.
`
`[00020]
`
`In another embodiment, the disclosure provides a pharmaceutical composition
`
`comprising a conditionally active biologic protein, and a pharmaceutically acceptable carrier.
`
`BRIEF DESCRIPTION OF THE DRAWING
`
`[00021]
`
`Figure 1 depicts a schematic representation of a chimeric antigen receptor in
`
`accordance with one embodiment of the present invention. ASTR is an antigen—specific
`
`targeting region, L is a linker, ESD is an extracellular spacer domain, TM is a transmembrane
`
`domain, CSD is a co—stimulatory domain, and ISD is an intracellular signaling domain.
`
`[00022]
`
`Figures 2 and 3 show that expressing the conditionally active antibodies of
`
`Example 7 as bivalent or monovalent antibodies does not significantly alter that selectivity of
`
`these antibodies under pH 6.0 and over pH 7.4.
`
`[00023]
`
`Figure 4 is a profile of a size exclusive chromatograph indicating that the
`
`conditionally active antibodies of Example 8 do not aggregate.
`
`[00024]
`
`Figure 5 shows on and off rates for the conditionally active antibodies of
`
`Example 8 as measured by a surface plasmon resonance (SPR) assay.
`
`[00025]
`
`Figures 6A—6B show the selectivity of the conditionally active antibodies as
`
`measured by the SPR assay of Example 8.
`
`

`

`WO 2016/036916
`
`PCT/U82015/048258
`
`DEFINITIONS
`
`[00026]
`
`In order to facilitate understanding of the examples provided herein, certain
`
`frequently occurring methods and/or terms will be defined herein.
`
`[00027]
`
`As used herein in connection with a measured quantity, the term "about" refers
`
`to the normal variation in that measured quantity that would be expected by the skilled artisan
`
`making the measurement and exercising a level of care commensurate with the objective of
`
`the measurement and the precision of the measuring equipment used. Unless otherwise
`
`indicated, "about" refers to a variation of +/— 10% of the value provided.
`
`[00028]
`
`The term "agent" is used herein to denote a chemical compound, a mixture of
`
`chemical compounds, an array of spatially localized compounds (e. g., a VLSIPS peptide
`
`array, polynucleotide array, and/or combinatorial small molecule array), biological
`
`macromolecule, a bacteriophage peptide display library, a bacteriophage antibody (e. g., scFv)
`
`display library, a polysome peptide display library, or an extract made from biological
`
`materials such as bacteria, plants, fungi, or animal (particular mammalian) cells or tissues.
`
`Agents are evaluated for potential enzyme activity by inclusion in screening assays described
`
`herein below. Agents are evaluated for potential activity as conditionally active biologic
`
`therapeutic enzymes by inclusion in screening assays described herein below.
`
`[00029]
`
`An "ambiguous base requirement" in a restriction site refers to a nucleotide
`
`base requirement that is not specified to the fullest extent, i.e. that is not a specific base (such
`
`as, in a non—limiting exemplification, a specific base selected from A, C, G, and T), but rather
`
`may be any one of at least two or more bases. Commonly accepted abbreviations that are
`
`used in the art as well as herein to represent ambiguity in bases include the following: R=G or
`
`A; Y=C or T; M=A or C; K=G or T; S=G or C; W=A or T; H=A or C or T; B=G or T or C;
`
`V=G or C or A; D=G or A or T; N=A or C or G or T.
`
`[00030]
`
`The term "amino acid" as used herein refers to any organic compound that
`
`contains an amino group (——NH2) and a carboxyl group (——COOH); preferably either as free
`
`groups or alternatively after condensation as part of peptide bonds. The "twenty naturally
`
`encoded polypeptide—forming alpha—amino acids" are understood in the art and refer to:
`
`alanine (ala or A), arginine (arg or R), asparagine (asn or N), aspartic acid (asp or D),
`
`cysteine (cys or C), gluatamic acid (glu or E), glutamine (gin or Q), glycine (gly or G),
`
`histidine (his or H), isoleucine (ile or I), leucine (leu or L), lysine (lys or K), methionine (met
`
`or M), phenylalanine (phe or F), proline (pro or P), serine (ser or S), threonine (thr or T),
`
`tryptophan (tip or W), tyrosine (tyr or Y), and valine (val or V).
`
`

`

`WO 2016/036916
`
`PCT/U82015/048258
`
`[00031]
`
`The term "amplification" means that the number of copies of a polynucleotide
`
`is increased.
`
`[00032]
`
`The term “antibody”, as used herein, refers to intact immunoglobulin
`
`molecules, as well as fragments of immunoglobulin molecules, such as Fab, Fab', (Fab')2, Fv,
`
`and SCA fragments, that are capable of binding to an epitope of an antigen. These antibody
`
`fragments, which retain some ability to selectively bind to an antigen (e.g., a polypeptide
`
`antigen) of the antibody from which they are derived, can be made using well known
`
`methods in the art (see, e. g., Harlow and Lane, supra), and are described further, as follows.
`
`Antibodies can be used to isolate preparative quantities of the antigen by immunoaffinity
`
`chromatography. Various other uses of such antibodies are to diagnose and/or stage disease
`
`(e.g., neoplasia) and for therapeutic application to treat disease, such as for example:
`
`neoplasia, autoimmune disease, AIDS, cardiovascular disease, infections, and the like.
`
`Chimeric, human—like, humanized or fully human antibodies are particularly useful for
`
`administration to human patients.
`
`[00033]
`
`An Fab fragment consists of a monovalent antigen—binding fragment of an
`
`antibody molecule, and can be produced by digestion of a whole antibody molecule with the
`
`enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy
`
`chain.
`
`[00034]
`
`An Fab' fragment of an antibody molecule can be obtained by treating a whole
`
`antibody molecule with pepsin, followed by reduction, to yield a molecule consisting of an
`
`intact light chain and a portion of a heavy chain. Two Fab‘ fragments are obtained per
`
`antibody molecule treated in this manner.
`
`[00035]
`
`An (Fab')2 fragment of an antibody can be obtained by treating a whole
`
`antibody molecule with the enzyme pepsin, without subsequent reduction. A (Fab')2 fragment
`
`is a dimer of two Fab' fragments, held together by two disulfide bonds.
`
`[00036]
`
`An FV fragment is defined as a genetically engineered fragment containing the
`
`variable region of a light chain and the variable region of a heavy chain expressed as two
`
`chains.
`
`[00037]
`
`The term “blood—brain barrier” or “BBB” refers to the physiological barrier
`
`between the peripheral circulation and the brain and spinal cord which is formed by tight
`
`junctions within the brain capillary endothelial plasma membranes, creating a tight barrier
`
`that restricts the transport of molecules into the brain, even very small molecules such as urea
`
`(60 Daltons). The blood—brain barrier within the brain, the blood—spinal cord barrier within
`
`the spinal cord, and the blood—retinal barrier within the retina are contiguous capillary barriers
`
`6
`
`

`

`WO 2016/036916
`
`PCT/U82015/048258
`
`within the central nerve system (CNS), and are herein collectively referred to as the “blood—
`
`brain barrier” or “BBB.” The BBB also encompasses the blood—cerebral spinal fluid barrier
`
`(choroid plexus) where the barrier is comprised of ependymal cells rather than capillary
`
`endothelial cells.
`
`[00038]
`
`The term “cell production host”, or “manufacturing host”, refers to a cell line
`
`used for the production or manufacturing of proteins. Eukaryotic cell production hosts such
`
`as mammalian cells, including, but not limited to human, mouse, hamster, rat, monkey cell
`
`lines as well as yeast, insect and plant cell lines. Prokaryotic cell production hosts can
`
`alternatively be utilized. In one aspect, a mammalian cell production host is selected from a
`
`member of the group consisting of 3T3 mouse fibroblast cells; BHK21 Syrian hamster
`
`fibroblast cells; MDCK, dog epithelial cells; Hela human epithelial cells; PtKl rat kangaroo
`
`epithelial cells; SP2/0 mouse plasma cells; and N80 mouse mouse plasma cells; HEK 293
`
`human embryonic kidney cells; COS monkey kidney cells; CHO, CHO—S Chinese hamster
`
`ovary cells; Rl mouse embryonic cells; El4.l mouse embryonic cells; H1 human embryonic
`
`cells; H9 human embryonic cells; PER C.6, human embryonic cells. In another aspect, the
`
`eukaryotic cell production host is a GS—NSO or GS—CHOKl cell line. In another aspect, the
`
`eukaryotic cell production host is selected from S. cerevisiae yeast cells; and picchia yeast
`
`cells. In another aspect, the cell production host is a bacterial cell line.
`
`[00039]
`
`The terms "cancer" and "cancerous" as used herein refer to or describe the
`
`physiological condition in mammals that is typically characterized by unregulated cell
`
`growth. Examples of cancer include, but are not limited to B—cell lymphomas (Hodgkin's
`
`lymphomas and/or non—Hodgkins lymphomas), brain tumor, breast cancer, colon cancer, lung
`
`cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian
`
`cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer,
`
`carcinoma, melanoma, head and neck cancer, brain cancer, and prostate cancer, including but
`
`not limited to androgen—dependent prostate cancer and androgen—independent prostate cancer.
`
`[00040]
`
`The term "chimeric antigen receptor" or "CAR" or "CARs" as used herein
`
`refers to engineered receptors, which graft an antigen specificity onto cytotoxic cell, for
`
`example T cells, NK cells and macrophages. The CARs of the invention may comprise at
`
`least one antigen specific targeting region (ASTR), an extracellular spacer domain (ESD), a
`
`transmembrane domain (TM), one or more co—stimulatory domains (CSD), and an
`
`intracellular signaling domain (ISD). In an embodiment, the ESD and/or CSD are optional. In
`
`another embodiment, the CAR is a bispecific CAR, which is specific to two different antigens
`
`or epitopes. After the ASTR binds specifically to a target antigen, the ISD activates
`
`7
`
`

`

`WO 2016/036916
`
`PCT/U82015/048258
`
`intracellular signaling. For example, the ISD can redirect T cell specificity and reactivity
`
`toward a selected target in a non—MHC—restricted manner, exploiting the antigen—binding
`
`properties of antibodies. The non—MHC—restricted antigen recognition gives T cells
`
`expressing the CAR the ability to recognize an antigen independent of antigen processing,
`
`thus bypassing a major mechanism of tumor escape. Moreover, when expressed in T cells,
`
`CARs advantageously do not dimerize with endogenous T cell receptor (TCR) alpha and beta
`
`chains.
`
`[00041]
`
`A molecule that has a "chimeric property" is a molecule that is: 1) in part
`
`homologous and in part heterologous to a first reference molecule; while 2) at the same time
`
`being in part homologous and in part heterologous to a second reference molecule; without 3)
`
`precluding the possibility of being at the same time in part homologous and in part
`
`heterologous to still one or more additional reference molecules. In a non—limiting
`
`embodiment, a chimeric molecule may be prepared by assembling a reassortment of partial
`
`molecular sequences. In a non—limiting aspect, a chimeric polynucleotide molecule may be
`
`prepared by synthesizing the chimeric polynucleotide using plurality of molecular templates,
`
`such that the resultant, chimeric polynucleotide has properties of a plurality of templates.
`
`[00042]
`
`The term "cognate" as used herein refers to a gene sequence that is
`
`evolutionarily and functionally related between species. For example, but not limitation, in
`
`the human genome the human CD4 gene is the cognate gene to the mouse 3d4 gene, since the
`
`sequences and structures of these two genes indicate that they are highly homologous and
`
`both genes encode a protein which functions in signaling T cell activation through MHC class
`
`H—restricted antigen recognition.
`
`[00043]
`
`The term “commercial scale” means production of a protein or antibody at a
`
`scale appropriate for resale.
`
`[00044]
`
`A "comparison window, " as used herein, refers to a conceptual segment of at
`
`least 20 contiguous nucleotide positions wherein a polynucleotide sequence may be
`
`compared to a reference sequence of at least 20 contiguous nucleotides and wherein the
`
`portion of the polynucleotide sequence in the comparison window may comprise additions or
`
`deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does
`
`not comprise additions or deletions) for optimal alignment of the two sequences. Optimal
`
`alignment of sequences for aligning a comparison window may be conducted by the local
`
`homology algorithm of Smith (Smith and Waterman, 1981 /'Comparison of biosequences",
`
`Adv Appl Math, 2:482—489; Smith and Waterman, 1981, "Overlapping genes and information
`
`theory", J Theor Biol,9l :3 79—3 80; Smith and Waterman, J Mol Biol, "Identification of
`
`8
`
`

`

`WO 2016/036916
`
`PCT/U82015/048258
`
`common molecular subsequences", 1981, 147: 195—197; Smith et al., 1981, ""Comparative
`
`biosequence metrics", J Mol Evol, 18:38—46), by the homology alignment algorithm of
`
`Needleman (Needleman and Wunsch, 1970, "A general method applicable to the search for
`
`similarities in the amino acid sequence oftwo proteins" JMOZ Biol, 48(3):443—453), by the
`
`search of similarity method of Pearson (Pearson and Lipman, 1988, "Improved tools for
`
`biological sequence comparison", Pmc Nat Acad Sci USA, 85:2444—2448), by computerized
`
`implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the
`
`Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science
`
`Dr., Madison, Wis), or by inspection, and the best alignment (i.e., resulting in the highest
`
`percentage of homology over the comparison window) generated by the various methods is
`
`selected.
`
`[00045]
`
`The term "conditionally active biologic protein" refers to a variant, or mutant,
`
`of a wild—type protein which is more or less active than the parent wild—type protein under
`
`one or more normal physiological conditions. This conditionally active protein also exhibits
`
`activity in selected regions of the body and/or exhibits increased or decreased activity under
`
`aberrant, or permissive, physiological conditions. Normal physiological conditions are those
`
`of temperature, pH, osmotic pressure, osmolality, oxidative stress and electrolyte
`
`concentration which would be considered within a normal range at the site of administration,
`
`or at the tissue or organ at the site of action, to a subject. An aberrant condition is that which
`
`deviates from the normally acceptable range for that condition. In one aspect, the
`
`conditionally active biologic protein is virtually inactive at wild—type conditions but is active
`
`at other than wild—type conditions at a level that is equal or better than at wild—type
`
`conditions. For example, in one aspect, an evolved conditionally active biologic protein is
`
`virtually inactive at body temperature, but is active at lower temperatures. In another aspect,
`
`the conditionally active biologic protein is reversibly or irreversibly inactivated at the wild
`
`type conditions. In a further aspect, the wild—type protein is a therapeutic protein. In another
`
`aspect, the conditionally active biologic protein is used as a drug, or therapeutic agent. In yet
`
`another aspect, the protein is more or less active in highly oxygenated blood, such as, for
`
`example, after passage through the lung or in the lower pH environments found in the kidney.
`
`[00046]
`
`"Conservative amino acid substitutions" refer to the interchangeability of
`
`residues having similar side chains. For example, a group of amino acids having aliphatic
`
`side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having
`
`aliphatic—hydroxyl side chains is serine and threonine; a group of amino acids having amide—
`
`containing side chains is asparagine and glutamine; a group of amino acids having aromatic
`
`9
`
`

`

`WO 2016/036916
`
`PCT/U82015/048258
`
`side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic
`
`side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur—
`
`containing side chains is cysteine and methionine. Preferred conservative amino acids
`
`substitution groups are: valine—leucine—isoleucine, phenylalanine—tyrosine, lysine—arginine,
`
`alanine—valine, and asparagine—glutamine.
`
`[00047]
`
`The term "corresponds to" is used herein to mean that a polynucleotide
`
`sequence is homologous (i.e., is identical, not strictly evolutionarily related) to all or a portion
`
`of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a
`
`reference polypeptide sequence. In contradistinction, the term "complementary to" is used
`
`herein to mean that the complementary sequence is homologous to all or a portion of a
`
`reference polynucleotide sequence. For illustration, the nucleotide sequence "TATAC"
`
`corresponds to a reference "TATAC" and is complementary to a reference sequence
`
`"GTATA."
`
`[00048]
`
`The term “co—stimulatory ligand” as used herein includes a molecule on an
`
`antigen presenting cell (e. g., dendritic cell, B cell, and the like) that specifically binds a
`
`cognate co—stimulatory molecule on a T cell, thereby providing a signal which, in addition to
`
`the primary signal provided by, for instance, by the binding of a TCIUCD3 complex With an
`
`MHC molecule loaded With peptide, mediates a T cell response, including, but not limited to,
`
`proliferation, activation, differentiation, and the like. A co—stimulatory ligand can include, but
`
`is not limited to, CD7, B7—1 (CD80), B7-2 (CD86), PD-Ll, PD—L2, 4-1BBL, OX40L, an
`
`inducible costimulatory ligand (ICOS—L), an intercellular adhesion molecule (ICAM),
`
`CD30L, CD40, CD70, CD83, HLA—G, MICA, MICB, HVEM, a lymphotoxin beta receptor,
`
`3/TR6, ILT3, ILT4, HVEM, an agonist or an antibody that binds to a Toll ligand receptor and
`
`a ligand that specifically binds with B7—H3. A co—stimulatory ligand also encompasses, inter
`
`alia, an antibody that specifically binds with a co—stimulatory molecule present on a T cell,
`
`such as, but not limited to, CD27, CD28, 4—lBB, 0X40, CD30, CD40, PD—l, ICOS, a
`
`lymphocyte function—associated antigen—l (LFA—l), CD2, CD7, LIGHT, NKG2C, B7—H3,
`
`and a ligand that specifically binds with CD83.
`
`[00049]
`
`The term “co—stimulatory molecule” as used herein refers to the cognate binding
`
`partner on a T cell that specifically binds with a co—stimulatory ligand, thereby mediating a
`
`co—stimulatory response by the T cell, such as, but not limited to, proliferation. Co—
`
`stimulatory molecules include, but are not limited to an MHC class 1 molecule, BTLA and a
`
`Toll ligand receptor.
`
`10
`
`

`

`WO 2016/036916
`
`PCT/U82015/048258
`
`[00050]
`
`The term “co—stimulatory signal” as used herein refers to a signal, which in
`
`combination with a primary signal, such as TCR/CD3 ligation, leads to T cell proliferation
`
`and/or upregulation or down regulation of key molecules.
`
`[00051]
`
`The term “cytotoxic cell” as used herein means a cell which can injure or
`
`destroy invading microorganisms, tumor cells or other diseased tissue cells. This term is
`
`meant to include natural killer (NK) cells, activated NK cells, neutrophils, T cells,
`
`eosinophils, basophils, B— cells, macrophages and lymphokine—activated killer (LAK) cells
`
`among other cell types. The cytotoxic cell, through an antibody, receptor, ligand or
`
`fragments/derivatives thereof, is bound to a target cell to form a stable complex, and
`
`stimulates the cytotoxic cell to destroy the target cell.
`
`[00052]
`
`The term "degrading effective" amount refers to the amount of enzyme which
`
`is required to process at least 50% of the substrate, as compared to substrate not contacted
`
`with the enzyme.
`
`[00053]
`
`As used herein, the term "defined sequence framework" refers to a set of
`
`defined sequences that are selected on a non—random basis, generally on the basis of
`
`experimental data or s