(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(19) World Intellectual Property
`Organization
`International Bureau
`
`(43) International Publication Date
`2 March 2017 (02.03.2017)
`
`WIPOI PCT
`
`\9
`
`(10) International Publication Number
`
`WO 2017/034615 A1
`
`(51)
`
`International Patent Classification:
`C07K 14/725 (2006.01)
`C12N 15/85 (2006.01)
`C07K 16/28 (2006.01)
`A61K 39/395 (2006.01)
`C07K 19/00 (2006.01)
`A61P 35/00 (2006.01)
`C12N 5/078 (2010.01)
`
`(21)
`
`International Application Number:
`
`PCT/US2016/019255
`
`(22)
`
`International Filing Date:
`
`24 February 2016 (24.02.2016)
`
`(25)
`
`(26)
`
`(30)
`
`(71)
`
`(72)
`
`(74)
`
`Filing Language:
`
`Publication Language:
`
`Priority Data:
`PCT/US2015/047197
`27 August 2015 (27.08.2015)
`
`English
`
`English
`
`US
`
`Applicant: BIOATLA, LLC [US/US];
`Road, San Diego, California 92121 (US).
`
`l 101 l Torreyana
`
`Inventor: SHORT, Jay M.; 12985 Via Esperia, Del Mar,
`California 92014 (US).
`
`Agent: DUNLEAVY, Kevin J.; Mendelsohn Dunleavy, P
`C, 1500 John F. Kennedy Blvd., Suite 312, Philadelphia,
`Pennsylvania 19102 (US).
`
`(81)
`
`Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY,
`BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM,
`DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`HN, HR, HU, 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:
`
`as to the identity ofthe inventor (Rule 4.1 7(2))
`
`as to applicant's entitlement to apply for and be granted a
`patent (Rule 4.1 7(ii))
`
`(54) Title: CONDITIONALLY ACTIVE CHIMERIC ANTIGEN RECEPTORS FOR MODIFIED T-CELLS
`
`[Continued on nextpage]
`
`(57) Abstract: This disclosure relates to a chimeric
`antigen receptor for binding With a target antigen. The
`chimeric antigen receptor comprises at least one anti-
`gen specific targeting region including a multispecific
`antibody evolved from a wild-type antibody or a frag-
`ment thereof and having at least one of: (a) a decrease
`in activity in the assay at the normal physiological
`condition compared to the Wild-type antibody or the
`fragment thereof, and (b) an increase in activity in the
`assay under the aberrant condition compared to the
`wild-type antibody or the fi‘agment thereof. A method
`for using the chimeric antigen receptor and cytotoxic
`cells for cancer treatment is also provided.
`
`
`
`bit/216111.
`
`“it
`
`monovalenl.
`
`CAB—SCFV Affinity ELESA
`
`OD450
`
`
`
`a pH 6.43
`
`m prim
`
`FIG. 2
`
`
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`WO 2017/034615 A1 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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`— as to the applicant’s entitlement to claim the priority of Published:
`the earlier application (Rule 4.17(iii))
`— with international search report (Art. 21(3))
`
`

`

`WO 2017/034615
`
`PCT/U82016/019255
`
`CONDITIONALLY ACTIVE CHIMERIC ANTIGEN RECEPTORS FOR MODIFIED
`
`T-CELLS
`
`FIELD OF THE DISCLOSURE
`
`[0001]
`
`This disclosure relates to the field of protein evolution. Specifically, this disclosure
`
`relates to a method of generating a conditionally active chimeric antigen receptor from a Wild
`
`type protein. The conditionally active chimeric antigen receptor is reversibly or irreversibly
`
`inactivated at a wild type normal physiological condition, but is active at an aberrant condition.
`
`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, enzymes may be
`
`evolved to be stabilized for operation at different conditions such as at an elevated temperature.
`
`In situations Where there is an activity improvement at the elevated 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.
`
`[0003]
`
`In addition, there exist examples of natural mutations that destabilize proteins at
`
`their normal operating conditions. Certain mutants can be active at a lower temperature, but at a
`
`reduced level compared to the Wild type proteins. This is also typically described by a reduction
`
`in activity as guided by the Q10 or similar rules.
`
`[0004]
`
`It is desirable to generate useful molecules that are conditionally activated. For
`
`example, it is desirable to generate molecules that are virtually inactive at Wild—type operating
`
`conditions but are active at other than wild-type operating conditions at a level that is equal to or
`
`better than at wild—type operating conditions, 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.
`
`[0005]
`
`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 preferably less than 1% activity) at a Wild type operating condition, While
`
`maintaining activity equivalent or better than its corresponding Wild type protein at a condition
`
`other than a wild—type operating condition, requires that destabilizing mutation(s) co—exist with
`
`activity increasing mutations that do not counter the destabilizing effect. It is expected that
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`WO 2017/034615
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`PCT/U82016/019255
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`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 the normal operating condition for the
`
`corresponding wild—type protein, creates an unexpected new class of proteins.
`
`[0006]
`
`Chimeric antigen receptors (CARs) have been used in treating cancers. US
`
`2013/0280220 discloses methods and compositions providing improved cells encoding a
`
`chimeric antigen receptor that is specific for two or more antigens, including tumor antigens.
`
`Cells expressing the chimeric antigen receptor may be used in cell therapy. Such cell therapy
`
`may be suitable for any medical condition, although in specific embodiments the cell therapy is
`
`for cancer, including cancer involving solid tumors.
`
`[0007]
`
`The present invention provides engineered conditionally active chimeric antigen
`
`receptors that are inactive or less active at a normal physiological condition but active at an
`
`aberrant physiological condition.
`
`[0008]
`
`Throughout this application, various publications are referenced by author and date.
`
`The disclosures of these publications are hereby incorporated by reference in their entireties 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.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`[0009]
`
`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.
`
`[00010]
`
`Figures 2 and 3 show that expressing the conditionally active antibodies of
`
`Example 1 as bivalent or monovalent antibodies does not significantly alter that selectivity of
`
`these antibodies under pH 6.0 and over pH 7.4.
`
`[00011]
`
`Figure 4 is a profile of a size exclusive chromatograph indicating that the
`
`conditionally active antibodies of Example 2 do not aggregate.
`
`[00012]
`
`Figure 5 shows on and off rates for the conditionally active antibodies of Example
`
`2 as measured by a surface plasmon resonance (SPR) assay.
`
`[00013]
`
`Figures 6A-6B show the selectivity of the conditionally active antibodies as
`
`measured by the SPR assay of Example 2.
`
`[00014]
`
`Figure 7A shows that CAR—T cells had no effect on a population of CHO cells that
`
`do not express the target antigen Xl of the CAR—T cells. The CAR molecule in the CAR—T cells
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`WO 2017/034615
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`PCT/U82016/019255
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`of this example included an antibody against target antigen X1, though this antibody was not
`
`conditionally active (Comparative Example A).
`
`[00015]
`
`Figure 7B shows that CAR—T cells reduced the population of CHO-63 cells that
`
`express the target antigen Xl of the CAR—T cells. These CAR—T cells are the same cells as were
`
`used to generate the data shown in Figure 7A (Comparative Example A).
`
`[00016]
`
`Figure 8A shows that CAR—T cells had no effect on a population of CHO cells that
`
`do not express the target antigen X1 of the CAR-T cells. The CAR molecule in the CAR-T cells
`
`of this Example 3 included a conditionally active antibody against target antigen Xl.
`
`[00017]
`
`Figure 8B shows that CAR—T cells reduced the population of CHO-63 cells that
`
`express the target antigen X1 of the CAR-T cells as tested in Example 3. These CAR-T cells are
`
`the same cells as were used to generate the data shown in Figure SA.
`
`[00018]
`
`Figures 9A-9B show cytokine release induced by binding of CAR-T cells with the
`
`target antigen X1, as described in Example 3.
`
`[00019]
`
`Figure 10 shows conditionally active antibodies against target antigen X2.
`
`[00020]
`
`Figure 1 1A shows the cytotoxic effect induced by CAR-T cells binding to Daudi
`
`cells that express target antigen X2 and the cytotoxic effect induced by CAR—T cells on HEK293
`
`cells that do not express target antigen X2, as described in Example 4.
`
`[00021]
`
`Figures 12A—12B show cytokine release induced by binding of CAR-T cells with
`
`the target antigen X1, as described in Example 5.
`
`[00022]
`
`Figures 13A—13B show cytokine release induced by binding of CAR-T cells with
`
`the target antigen X2, as described in Example 5.
`
`[00023]
`
`Figure 14 shows conditionally active antibodies against target antigen X3 that are
`
`suitable for construction of CAR—T cells.
`
`SUMMARY OF THE DISCLOSURE
`
`[00024]
`
`In one aspect, the present invention provides a chimeric antigen receptor (CAR) for
`
`binding with a target antigen. The CAR includes at least one antigen specific targeting region.
`
`The CAR is an antibody evolved from a wild-type antibody or a fragment thereof. The CAR
`
`includes a transmembrane domain; an intracellular signaling domain; and has at least one of: (a)
`
`a decrease in binding activity to the target antigen in an assay at a normal physiological
`
`condition compared to the wild—type antibody or a fragment thereof, and (b) an increase in
`
`binding activity to the target antigen in an assay at an aberrant condition compared to the wild-
`
`type antibody or a fragment thereof. The normal and aberrant conditions are a same condition
`
`having different values.
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`

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`WO 2017/034615
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`PCT/U82016/019255
`
`[00025]
`
`In yet another aspect, the present invention provides a chimeric antigen receptor for
`
`binding with a target antigen. The CAR includes at least one antigen specific targeting region
`
`evolved from a wild—type protein or a domain thereof. The CAR also has at least one of: (a) a
`
`decrease in activity in an assay at a normal physiological condition compared to the antigen
`
`specific targeting region of the wild-type protein or a domain thereof, and (b) an increase in
`
`activity in an assay under an aberrant condition compared to the antigen specific targeting region
`
`of the wild—type protein or a domain thereof. The normal and aberrant conditions are a same
`
`condition having different values. The aberrant condition is a condition present in a tumor
`
`microenvironment, a brain extracellular fluid, a stem cell niche, a lymph node, a tonsil, an
`
`adenoid, a sinus, or a synovial fluid. The CAR further comprises a transmembrane domain and
`
`an intracellular signaling domain.
`
`[00026]
`
`In yet another aspect, the present invention provides a chimeric antigen receptor for
`
`binding with a target antigen. The CAR includes a transmembrane domain; an intracellular
`
`signaling domain and at least two antigen specific targeting regions connected by a conditional
`
`linker. The conditional linker has a first conformation at an aberrant condition that presents the
`
`at least two antigen specific targeting regions in a manner which has a higher binding affinity to
`
`the target antigen than a binding affinity of a second conformation of the same conditional linker
`
`that occurs at a normal physiological condition.
`
`[00027]
`
`In yet another aspect, the chimeric antigen receptor has one or both of two antigen
`
`specific targeting regions evolved from a wild-type protein or a domain thereof. The CAR also
`
`has at least one of: (a) a decrease in activity in an assay at a normal physiological condition
`
`compared to the antigen specific targeting region of the wild—type protein or a domain thereof,
`
`and (b) an increase in activity in an assay under an aberrant condition compared to the antigen
`
`specific targeting region of the wild-type protein or a domain thereof.
`
`[00028]
`
`In yet another aspect, the present invention provides a chimeric antigen receptor for
`
`binding with a target antigen The CAR includes a transmembrane domain; an intracellular
`
`signaling domain; and at least one antigen specific targeting region that binds with the target
`
`antigen; an extracellular spacer domain having a first conformation at an aberrant condition for
`
`the at least one antigen specific targeting region that provides a higher binding affinity to the
`
`target antigen than a second conformation of the antigen specific extracellular spacer domain
`
`that occurs at a normal physiological condition.
`
`[00029]
`
`In yet another aspect, the chimeric antigen receptor has an antigen specific
`
`targeting region evolved from a wild—type protein or a domain thereof. The CAR also has at
`
`least one of: (a) a decrease in activity in an assay at a normal physiological condition compared
`
`to the antigen specific targeting region of the wild-type protein or a domain thereof, and (b) an
`4
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`WO 2017/034615
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`PCT/U82016/019255
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`increase in activity in an assay under an aberrant condition compared to the antigen specific
`
`targeting region of the wild—type protein or a domain thereof. The normal and aberrant
`
`conditions are a same condition having different values.
`
`[00030]
`
`In yet another aspect, the present invention provides a chimeric antigen receptor for
`
`binding with a target antigen. The CAR includes at least one antigen specific targeting region
`
`that binds with the target antigen; an extracellular spacer domain having an enhanced
`
`ubiquitylation-resistance at an aberrant condition than at a normal physiological condition; a
`
`transmembrane domain; and an intracellular signaling domain.
`
`[00031]
`
`In another aspect, the present invention provides an expression vector, including a
`
`polynucleotide sequence encoding the chimeric antigen receptor of the invention. The
`
`expression vector is selected from lentivirus vectors, gamma retrovirus vectors, foamy virus
`
`vectors, adeno associated virus vectors, adenovirus vectors, pox virus vectors, herpes virus
`
`vectors, engineered hybrid viruses, and transposon mediated vectors.
`
`[00032]
`
`In yet another aspect, the present invention provides a genetically engineered
`
`cytotoxic cell that includes a polynucleotide sequence encoding the chimeric antigen receptor of
`
`the invention. The cytotoxic cell may be a T cell and may be selected from a naive T cell, a
`
`central memory T cell, and an effector memory T cell.
`
`[00033]
`
`In yet another aspect, the present invention provides a pharmaceutical composition,
`
`including the chimeric antigen receptor, the expression vector, and/or the genetically engineered
`
`cytotoxic cell of the invention, and a pharmaceutically acceptable excipient.
`
`[00034]
`
`In yet another aspect, the present invention provides a method for treating a cancer
`
`in a subject, including the step of introducing an expression vector including a polynucleotide
`
`sequence encoding the chimeric antigen receptor of the invention into a cytotoxic cell obtained
`
`from the subject to produce a genetically engineered cytotoxic cell; and administering the
`
`genetically engineered cytotoxic cell to the subject.
`
`DEFINITIONS
`
`[00035]
`
`In order to facilitate understanding of the examples provided herein, certain
`
`frequently occurring methods and/or terms Will be defined herein.
`
`[00036]
`
`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.
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`WO 2017/034615
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`PCT/U82016/019255
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`[00037]
`
`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.
`
`[00038]
`
`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 1), 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).
`
`[00039]
`
`The term "amplification" as used herein means that the number of copies of a
`
`polynucleotide is increased.
`
`[00040]
`
`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.
`
`[00041]
`
`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.
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`[00042]
`
`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.
`
`[00043]
`
`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.
`
`[00044]
`
`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.
`
`[00045]
`
`The term “antigen” or “Ag” as used herein is defined as a molecule that provokes
`
`an immune response. This immune response may involve either antibody production, or the
`
`activation of specific immunologically—competent cells, or both. A person skilled in the art will
`
`understand that any macromolecule, including virtually all proteins or peptides, can serve as an
`
`antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A person
`
`skilled in the art will understand that any DNA, which includes a nucleotide sequence or a
`
`partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes
`
`an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that
`
`an antigen need not be encoded solely by a full length nucleotide sequence of a gene. It is
`
`readily apparent that the present invention includes, but is not limited to, the use of partial
`
`nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in
`
`various combinations to elicit the desired immune response. Moreover, a skilled person will
`
`understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an
`
`antigen can be generated, synthesized or can be derived from a biological sample. Such a
`
`biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a
`
`biological fluid.
`
`[00046]
`
`"Antigen loss escape variants" as used herein refer to cells which exhibit reduced or
`
`loss of expression of the target antigen, which antigens are targeted by the CARs of the
`
`invention.
`
`[00047]
`
`The term “autoimmune disease” as used herein is defined as a disorder that results
`
`from an autoimmune response. An autoimmune disease is the result of an inappropriate and
`
`excessive response to a self—antigen. Examples of autoimmune diseases include but are not
`
`limited to, Addison's disease, alopecia greata, ankylosing spondylitis, autoimmune hepatitis,
`
`autoimmune parotitis, Crohn's disease, diabetes (Type 1), dystrophic epidermolysis bullosa,
`
`epididymitis, glomerulonephritis, Graves' disease, Guillain—Barr syndrome, Hashimoto's disease,
`
`hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis,
`7
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`pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma,
`
`Sjogren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema,
`
`pernicious anemia, ulcerative colitis, among others.
`
`[00048]
`
`The term “autologous,” as used herein refers to any material derived from the same
`
`individual to which it is later to be reintroduced. For example, T cells from a patient may be
`
`isolated, genetically engineered to express a CAR and then reintroduced into the patient.
`
`[00049]
`
`The term ”B—cell associated diseases” as used herein include B-cell
`
`immunodeficiencies, autoimmune diseases and/or excessive/uncontrolled cell proliferation
`
`associated with B- cells (including lymphomas and/or leukemia’s). Examples of such diseases,
`
`wherein bispecific CARS of the invention may be used for therapeutic approaches include but
`
`are not limited to systemic lupus erythematosus (SLE), diabetes, rheumatoid arthritis (R A),
`
`reactive arthritis, multiple sclerosis (MS), pemphigus vulgaris, celiac disease, Crohn‘s disease,
`
`inflammatory bowel disease, ulcerative colitis, autoimmune thyroid disease, X— linked
`
`agammaglobulinaemis, pre-B acute lymphoblastic leukemia, systemic lupus erythematosus,
`
`common variable immunodeficiency, chronic lymphocytic leukemia, diseases associated with
`
`selective lgA deficiency and/or lgG subclass deficiency, B lineage lymphomas (Hodgkin's
`
`lymphoma and/or non—Hodgkin's lymphoma), immunodeficiency with thymoma, transient
`
`hypogammaglobulinemia and/or hyper IgM syndrome, as well as virally-mediated B-cell
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`diseases such as EBV mediated lymphoproliferative disease, and chronic infections in which B—
`
`cells participate in the pathophysiology.
`
`[00050]
`
`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
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`cord, and the blood-retinal barrier within the retina are contiguous capillary barriers within the
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`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 included of ependymal cells rather than capillary endothelial cells.
`
`[00051]
`
`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
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`and/or non—Hodgkins lymphomas), brain tumor, breast cancer, colon cancer, lung cancer,
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`hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver
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`cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma,
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`melanoma, head and neck cancer, brain cancer, and prostate cancer, including but not limited to
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`androgen—dependent prostate cancer and androgen-independent prostate cancer.
`
`[00052]
`
`The term "chimeric antigen receptor" or "CAR” or "CARs” as used herein refers to
`
`engineered receptors, which graft antigen specificity onto a cytotoxic cell, for example T cells,
`
`NK cells and macrophages. The CARs of the invention may include 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 intracellular signaling. For example, the ISD can redirect T
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`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.
`
`[00053]
`
`The term "co—express” as used herein refers to simultaneous expression of two or
`
`more genes. Genes may be nucleic acids encoding, for example, a single protein or a chimeric
`
`protein as a single polypeptide chain. For example, the CARs of the invention may be co-
`
`expressed with a therapeutic control (for example truncated epidermal growth factor (EGFRt)),
`
`wherein the CAR is encoded by a first polynucleotide chain and the therapeutic control is
`
`encoded by a second polynucleotide chain. In an embodiment, the first and second
`
`polynucleotide chains are linked by a nucleic acid sequence that encodes a cleavable linker.
`
`Alternately, the CAR and the therapeutic control are encoded by two different polynucleotides
`
`that are not linked via a linker but are instead encoded by, for example, two different vectors.
`
`[00054]
`
`The term "cognate" as used herein refers to a gene sequence that is evolutionarily
`
`and functionally related between species. For example, but without 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 II—restricted
`
`antigen recognition.
`
`[00055]
`
`The tenn ”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
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`permissive, physiological conditions. The term “normal physiological condition” as used herein
`
`refers to one of temperature, pH, osmotic pressure, osmolality, oxidative stress, electrolyte
`
`concentration, a concentration of a small organic molecule such as glucose, lactic acid, pyruvate,
`
`nutrient components, other metabolites, and the like, a concentration of another molecule such
`
`as oxygen, carbonate, phosphate, and carbon dioxide, as well as cell types, and nutrient
`
`availability, 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.
`
`[00056]
`
`The term “aberrant condition” as used herein refers to a condition that deviates
`
`from the normally acceptable range for that condition. In one aspect, the conditionally active
`
`biologic protein is virtually inactive at a normal physiological condition but is active at an
`
`aberrant condition at a level that is equal or better than the wild-type protein from which it is
`
`derived. 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 normal
`
`physiological condition. 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.
`
`[00057]
`
`"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 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: val