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WO2022047417A1 - Compositions anti-idiotype et procédés d'utilisation associés - Google Patents

Compositions anti-idiotype et procédés d'utilisation associés Download PDF

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Publication number
WO2022047417A1
WO2022047417A1 PCT/US2021/048532 US2021048532W WO2022047417A1 WO 2022047417 A1 WO2022047417 A1 WO 2022047417A1 US 2021048532 W US2021048532 W US 2021048532W WO 2022047417 A1 WO2022047417 A1 WO 2022047417A1
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WIPO (PCT)
Prior art keywords
polypeptide
cell
polynucleotide
idiotype
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/US2021/048532
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English (en)
Inventor
Gregory Ian Frost
Gregory Harold SCHREIBER
Anirban Kundu
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Exuma Biotech Corp
Original Assignee
Exuma Biotech Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/US2020/048843 external-priority patent/WO2021042072A1/fr
Priority claimed from PCT/US2021/020922 external-priority patent/WO2021178701A1/fr
Priority to CN202180052913.6A priority Critical patent/CN116249559A/zh
Priority to EP21862987.1A priority patent/EP4204004A4/fr
Priority to US18/043,465 priority patent/US20230357436A1/en
Application filed by Exuma Biotech Corp filed Critical Exuma Biotech Corp
Priority to PCT/US2022/018404 priority patent/WO2022187289A1/fr
Priority to US17/684,405 priority patent/US20230044451A1/en
Priority to EP22715804.5A priority patent/EP4301862A1/fr
Priority to MX2023010059A priority patent/MX2023010059A/es
Priority to JP2023553055A priority patent/JP2024510933A/ja
Priority to AU2022229358A priority patent/AU2022229358A1/en
Priority to CA3212366A priority patent/CA3212366A1/fr
Publication of WO2022047417A1 publication Critical patent/WO2022047417A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

Definitions

  • PCT/US2017/023112 claims the benefit of U.S. Provisional Application No. 62/390,093, filed March 19, 2016; U.S. Provisional Application No. 62/360,041, filed July 8, 2016; and U.S. Provisional Application No. 62/467,039, filed March 3, 2017; International Application No. PCT/US2017/041277 claims the benefit of International Application No. PCT/US2017/023112, filed March 19, 2017; U.S. Patent Application No. 15/462,855, filed March 19, 2017; U.S. Provisional Application No. 62/360,041, filed July 8, 2016; and U.S. Provisional Application No. 62/467,039, filed March 3, 2017; U.S. Application No.
  • This disclosure relates to the field of immunology, or more specifically, to the genetic modification of T lymphocytes or other immune cells, and methods of controlling proliferation of such cells.
  • Lymphocytes from a subject can be genetically modified ex vivo or in vivo to express synthetic proteins that enable redirected engagement with other cells and environments based upon the genetic programs incorporated.
  • synthetic proteins include engineered T cell receptors (TCRs) and chimeric antigen receptors (CARs).
  • TCRs engineered T cell receptors
  • CARs chimeric antigen receptors
  • Such lymphocytes have become important therapeutics (e.g. CAR-T therapy) for treating diseases such as cancer.
  • EGFR epidermal growth factor receptor
  • Patent 8,802,374 or WO2018226897 are polypeptides that are recognized by an antibody that recognizes the extracellular domain of an EGFR member.
  • Such truncated EGFR polypeptides are sometimes referred to herein as eTags.
  • An eTag was demonstrated to have cell killing potential through Erbitux® mediated antibody dependent cellular cytotoxicity (ADCC) pathways.
  • ADCC antibody dependent cellular cytotoxicity
  • eTags include EGFR family member domains, and thus are limited in structure to only EGFR domains. Furthermore, they may yield undesirable side effects when delivered to a subject since they are structurally related to growth factors (e.g. EGFR) found in a subject.
  • growth factors e.g. EGFR
  • anti-idiotype polypeptide safety switches are much more robust and flexible safety switches than prior art safety switches, such as eTags, in terms of design and development.
  • Anti-idiotype polypeptide safety switches can be designed to be recognized by virtually any antibody, including any clinical antibody, such as a therapeutic antibody. Exemplary methods for developing anti-idiotype antibodies, are known.
  • provided herein are methods, compositions, and kits that are, or include or encode, anti-idiotype polypeptides. Furthermore, provided herein are methods for delivering modified lymphocytes, especially modified and genetically modified T cell and/or NK cells, and/or for regulating the activity of transduced, genetically modified, and/or modified T cells and/or NK cells.
  • Such methods, compositions, and kits provide improved efficacy and safety over current technologies, especially with respect to T cells and/or NK cells that express lymphoproliferative elements (e.g., chimeric cytokine receptors), engineered T cell receptors (TCRs), and chimeric antigen receptors (CARs), including microenvironment restricted biologic (“MRB”) CARs.
  • lymphoproliferative elements e.g., chimeric cytokine receptors
  • TCRs engineered T cell receptors
  • CARs chimeric antigen receptors
  • MRB microenvironment restricted biologic
  • Transduced and/or modified and in illustrative embodiments genetically modified T cells and/or NK cells that are produced by and/or used in methods provided herein include anti-idiotype functionality typically, in combination with other functionality, in illustrative embodiments delivered from retroviral (e.g., lentiviral) genomes via retroviral (e.g., lentiviral) particles.
  • the anti-idiotype polypeptides, or polynucleotides encoding the same provide improved features for such cells, including safety features and for methods that utilize such cells, such as research methods, commercial production methods, and adoptive cellular therapy.
  • such cells can be controllably killed if they become safety concerns to subjects in which they are delivered, and can have improved growth properties that can be better regulated.
  • a polynucleotide comprising one or more transcriptional units, wherein each of the one or more transcriptional units is operatively linked to a promoter wherein the one or more transcriptional units comprise: a) a polynucleotide sequence encoding one or more inhibitory RNA molecules and/or a first engineered signaling polypeptide, and b) a polynucleotide sequence encoding an anti-idiotype polypeptide comprising an extracellular recognition domain and a membrane association domain, wherein the extracellular recognition domain comprises a domain that recognizes an idiotype of a target antibody or a target antibody mimetic.
  • a method for administering a cell therapy to a mammalian subject comprising administering modified cells to the mammalian subject, wherein the modified cells each comprise a polynucleotide encoding: a) one or more inhibitory RNA molecules and/or a first engineered polypeptide, and b) an anti-idiotype polypeptide comprising an extracellular recognition domain and a membrane association domain, wherein the extracellular recognition domain comprises a domain that recognizes an idiotype of a target antibody or a target antibody mimetic.
  • a method for administering a gene therapy to a mammalian subject comprising administering gene vectors to the mammalian subject, wherein the gene vectors each comprise a polynucleotide encoding: a) one or more inhibitory RNA molecules and/or a first engineered polypeptide, and b) an anti-idiotype polypeptide comprising an extracellular recognition domain and a membrane association domain, wherein the extracellular recognition domain comprises a domain that recognizes an idiotype of a target antibody or a target antibody mimetic.
  • the anti-idiotype polypeptide is capable of binding to an idiotype of a clinical stage or approved target antibody or antibody mimetic.
  • target antibody or antibody mimetic can be capable of, adapted for, configured to, and/or effective for promoting cell death.
  • the anti-idiotype polypeptide further include one or more intracellular domains.
  • the intracellular signaling domains can activate or inhibit pro-apoptotic or anti-apoptotic pathways and/or pro-survival or anti-survival pathways.
  • FIG.l shows a non-limiting example of a polynucleotide that is a portion of a lenti viral vector, with nucleic acids that encode an anti-idiotype polypeptide includes an anti-idiotype extracellular recognition domain (“Anti-idiotype”), a stalk, and a membrane association domain (MAD).
  • the vector also includes a promoter (“Promoter”) positioned to drive expression of nucleic acids encoding different functional polypeptides including the anti-idiotype polypeptide, and includes various functional domains as indicated, including various viral functional domains, and encodes an inhibitory RNA molecule within the promoter.
  • Promoter positioned to drive expression of nucleic acids encoding different functional polypeptides including the anti-idiotype polypeptide, and includes various functional domains as indicated, including various viral functional domains, and encodes an inhibitory RNA molecule within the promoter.
  • FIGs. 2A-G show non-limiting examples of polynucleotides that include nucleic acids that encode one or more anti-idiotype polypeptides in various combinations with other polypeptides.
  • FIG. 2A shows nucleic acids that encode an anti-idiotype polypeptide with a membrane association domain (MAD) and an anti-idiotype extracellular recognition domain (referred to as “anti-idiotype” in FIG. 2).
  • FIGs. 2B-D show an anti-idiotype polypeptide expressed as part of a single polynucleotide that also encodes a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR) (FIG. 2B), a lymphoproliferative element (FIG.
  • CAR chimeric antigen receptor
  • TCR engineered T cell receptor
  • FIGs. 2E-G shows an anti-idiotype polypeptide is expressed as part of a single polynucleotide that also encodes a CAR or a TCR, an antiidiotype polypeptide, along with a lymphoproliferative element (FIG. 2E), or a cytokine (FIG. 2F), or both a lymphoproliferative element and a cytokine (FIG. 2G).
  • FIGs. 3A-G show non-limiting examples of anti-idiotype polypeptides.
  • FIG. 3A shows an antiidiotype polypeptide with an extracellular recognition domain (sometimes referred to as an anti-idiotype extracellular recognition domain, anti-id ERD, or anti-id ECD, and denoted in FIGs. 3A-F and FIGs. 3H as “anti-idiotype”), a stalk, and a membrane association domain (MAD).
  • FIGs. 3B-3G show anti-idiotype polypeptides with one or more intracellular domains (sometimes referred to herein as ICDs) and a transmembrane domain (“TM” in FIGs. 3B-3G).
  • ICDs intracellular domains
  • TM transmembrane domain
  • FIG. 3B shows the anti-idiotype polypeptide with a single intracellular domain.
  • FIGs. 3C-E shows the anti-idiotype polypeptide with either the P3 domain, and optionally a P4 domain, of a lymphoproliferative element (FIG. 3C), or with the intracellular domain of a CAR or TCR (FIG. 3D), or with the intracellular domain of a death domain.
  • FIG. 3F shows an antiidiotype polypeptide with the anti-id ERD as one of the ASTRs of a bi-specific CAR or TCR.
  • FIG. 3G shows an anti-idiotype polypeptide similar to FIG. 3B, but with a cleavage site between or as part of the transmembrane domain and the ICD.
  • FIG. 4 shows show non-limiting examples of anti-idiotype polypeptides.
  • FIGs. 5A and 5B show schematics of illustrative bicistronic lentiviral genomic vectors with divergent transcriptional units.
  • FIG. 6 A shows the panning round, phage input (pfu), bead volume, binding time, number of washes, phage output (pfu) recovery rate, and enrichment for Panning Experiment 1 using DynabeadsTM Protein A.
  • FIG 6B shows the panning round, phage input (pfu), bead volume, binding time, number of washes, phage output (pfu) recovery rate, and enrichment for Panning Experiment 1 using DynabeadsTM M-270 Epoxy
  • FIG. 7 shows the binding of the phage output from rounds 1-5 of Panning Experiment 1 to cetuximab as measured by ELISA. Phage were eluted from DynabeadsTM M-270 Epoxy using Elution Buffer in each panning round except as indicated for R5 in which bound phage were eluted with EGFR.
  • FIG. 8 shows sequence information for the 19 unique clones identified in Panning Experiment 1 using Ab 1.1 as bait
  • FIG. 9A-9C shows sequence information for the 42 unique clones identified in Panning Experiment 2 using Ab 1.2 as bait
  • FIG. 10 shows the results of a competitive binding assay in which soluble EGFR competed with monoclonal phage clones from Panning Experiment 2 for binding to cetuximab by ELISA.
  • FIG. 11 shows a schematic of an illustrative lentiviral genomic vector with a single transcriptional unit encoding a CAR and an anti-idiotype polypeptide separated by a ribosomal skip sequence.
  • FIG. 12 shows the expression of 2 different cetuximab anti-idiotype polypeptides on 293T and CHO-S cells as detected by cetuximab and assayed by FACs.
  • chimeric antigen receptor or “CAR” or “CARs” refers to engineered receptors, which graft an antigen specificity onto cells, for example T cells, NK cells, macrophages, and stem cells.
  • the CARs of the invention include at least one antigen-specific targeting region (ASTR), a transmembrane domain (TM), and an intracellular activating domain (I AD) and can include a stalk, and one or more co-stimulatory domains (CSDs).
  • ASTR antigen-specific targeting region
  • TM transmembrane domain
  • I AD intracellular activating domain
  • 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 IAD activates intracellular signaling.
  • the IAD 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.
  • CARs advantageously do not dimerize with endogenous T cell receptor (TCR) alpha and beta chains.
  • the term cell “aggregate” means a cluster of cells that adhere to each other.
  • the term “constitutive T cell or NK cell promoter” refers to a promoter which, when operably linked with a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.
  • inducible promoter or “activatable promoter” refer to promoters that when operably linked with a polynucleotide that encodes or specifies a gene product, cause the gene product to be produced in a cell substantially only when a promoter-specific inducer is present in the cell. Inducible promoters have no, or a low level, of basal transcription activity but the transcription activity increases, sometimes greatly, in the presence of an inducing signal.
  • insulator refers to a cis-regulatory element that mediates intra- and inter-chromosomal interactions and can block interactions between enhancers and promoters. Typically, insulators are between 200 and 2000 base pairs in length and contain clustered binding sites for sequence specific DNA-binding proteins.
  • the term "microenvironment” means any portion or region of a tissue or body that has constant or temporal, physical, or chemical differences from other regions of the tissue or regions of the body.
  • a tumor microenvironment refers to the environment in which a tumor exists, which is the non-cellular area within the tumor and the area directly outside the tumorous tissue but does not pertain to the intracellular compartment of the cancer cell itself.
  • the tumor microenvironment can refer to any and all conditions of the tumor milieu including conditions that create a structural and or functional environment for the malignant process to survive and/or expand and/or spread.
  • the tumor microenvironment can include alterations in conditions such as, but not limited to, pressure, temperature, pH, ionic strength, osmotic pressure, osmolality, oxidative stress, concentration of one or more solutes, concentration of electrolytes, concentration of glucose, concentration of hyaluronan, concentration of lactic acid or lactate, concentration of albumin, levels of adenosine, levels of R-2-hydroxy glutarate, concentration of pyruvate, concentration of oxygen, and/or presence of oxidants, reductants, or co-factors, as well as other conditions a skilled artisan will understand.
  • conditions such as, but not limited to, pressure, temperature, pH, ionic strength, osmotic pressure, osmolality, oxidative stress, concentration of one or more solutes, concentration of electrolytes, concentration of glucose, concentration of hyaluronan, concentration of lactic acid or lactate, concentration of albumin, levels of adenosine, levels of R-2-hydroxy gluta
  • polynucleotide and “nucleic acid” refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides.
  • this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
  • an “approved biologic” is a macromolecule that meets the requirements of a biologic provided by a government regulatory agency such as, but not limited to, the Food And Drug Administration of the U.S. (USFDA), European Medicines Agency (EMA), National Medical Products Administration of China (NMPA) (Chinese FDA), or the Pharmaceutical and Food Safety Bureau (PFSB) of Japan and has been approved by such regulatory agency either as a stand-alone biologic, or as part of a combination product or method.
  • USFDA Food And Drug Administration of the U.S.
  • EMA European Medicines Agency
  • NMPA National Medical Products Administration of China
  • PFSB Pharmaceutical and Food Safety Bureau
  • antibody includes polyclonal and monoclonal antibodies, including intact antibodies and fragments of antibodies which retain specific binding to antigen.
  • the antibody fragments can be, but are not limited to, fragment antigen binding (Fab) fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, Fab'-SH fragments, (Fab')2 Fv fragments, Fd fragments, recombinant IgG (rlgG) fragments, single-chain antibody fragments, including single-chain variable fragments (scFv), divalent scFv's, trivalent scFv's, and single domain antibody fragments (e.g., sdAb, sdFv, nanobody).
  • the term includes genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, single-chain antibodies, fully human antibodies, humanized antibodies, fusion proteins including an antigen-specific targeting region of an antibody and a nonantibody protein, heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv's, and tandem tri-scFv's.
  • antibody should be understood to include functional antibody fragments thereof.
  • the term also includes intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and subclasses thereof, IgM, IgE, IgA, and IgD.
  • antibody fragment includes a portion of an intact antibody, for example, the antigen binding or variable region of an intact antibody.
  • antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab” fragments, each with a single antigen-binding site, and a residual "Fe” fragment, a designation reflecting the ability to crystallize readily.
  • Pepsin treatment yields an F(ab')2 fragment that has two antigen combining sites and is still capable of cross-linking antigen.
  • the terms "single-chain Fv,” “scFv,” or “sFv” antibody fragments include the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further includes a polypeptide linker or spacer between the VH and VL domains, which enables the sFv to form the desired structure for antigen binding.
  • VH and VL domains refer to VH and VL domains that have been isolated from a host without further molecular evolution to change their affinities when generated in an scFv format under specific conditions such as those disclosed in US patent 8709755 B2 and application WO/2017/033331A1.
  • antibody mimetic refers to an organic compound that specifically binds a target sequence and has a structure distinct from a naturally-occurring antibody.
  • Antibody mimetics may comprise a protein, a nucleic acid, or a small molecule, and a skilled artisan can understand when each type is relevant.
  • the target sequence to which an antibody mimetic of the disclosure specifically binds may be an antigen.
  • Antibody mimetics may provide superior properties over antibodies including, but not limited to, superior solubility, tissue penetration, stability towards heat and enzymes (e.g., resistance to enzymatic degradation), and lower production costs.
  • Antibody mimetics include, but are not limited to, an affibody, an afflilin, an affimer, an affitin, an alphabody, an alphamab, an anticalin, a peptide aptamer, an armadillo repeat protein, an atrimer, an avimer (also known as avidity multimer), a C-type lectin domain, a cysteine-knot miniprotein, a cyclic peptide, a cytotoxic T-lymphocyte associated protein-4, a DARPin (Designed Ankyrin Repeat Protein), a fibrinogen domain, a fibronectin binding domain (FN3 domain) (e.g., adnectin or monobody), a fynomer, a knottin, a Kunitz domain peptide, a nanofitin, a leucine -rich repeat domain, a lipocalin domain, a mAb 2 or FcabTM, a nanobody, a nanoCL
  • CDR complementarity-determining region
  • the sequences of the framework regions of different light or heavy chains are relatively conserved within a species.
  • the framework region of an antibody that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three dimensional space.
  • the amino acid sequences of the CDRs and framework regions can be determined using various well-known definitions in the art, e.g., Kabat, Chothia, international ImMunoGeneTics database (IMGT), and AbM (see, e.g., Johnson and Wu, Nucleic Acids Res. 2000 Jan 1; 28(1): 214-218 and Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia & Lesk, (1987) J. Mol. Biol.
  • CDRs herein are determined using “Fab Analysis” on the World Wide Web at vbase2.org (Retter et al., Nucleic Acids Res., 33:D671-D674 and Mollova et al., BMC Systems Bio., SI, p30)).
  • idiotype refers to the segment of an antibody or antibody mimetic that determines its specificity for antigen, for example, a structure of a variable region of an antibody, T cell receptor, or antibody mimetic that is shared characteristic between a group of antibodies, T-cell receptors, or antibody mimetics based upon the antigen binding specificity and therefore structure of their variable regions.
  • the idiotype of an antibody typically includes the variable region, e.g., the CDRs and framework regions.
  • the idiotype is located in the Fab region.
  • expression of the idiotype usually requires participation of the variable regions of both heavy and light chains that form the antigen-combining site.
  • scFv For antibodies formed with single chains, e.g., scFv, expression of the idiotype usually requires participation of the variable regions of one polypeptide that forms the antigen-combining site.
  • the idiotype varies depending on the type of antibody mimetic, but includes the region necessary for binding the cognate antigen.
  • a “therapeutic antibody” or “therapeutic antibody mimetic” is an antibody or antibody mimetic that has been demonstrated using an in vivo assay, for example, in humans, to have therapeutic activity.
  • the term “recognize” refers to the ability of one molecule to bind to another molecule, for example, the ability of a receptor to binds its ligand or the ability of an antibody to bind its target.
  • affinity refers to the equilibrium constant for the reversible binding of two agents and is expressed as a dissociation constant (Kd).
  • Kd dissociation constant
  • Affinity can be at least 1-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20- fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-fold greater, or more, than the affinity of an antibody for unrelated amino acid sequences.
  • Affinity of an antibody to a target protein can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM) or more.
  • nM nanomolar
  • pM picomolar
  • fM femtomolar
  • the term “avidity” refers to the resistance of a complex of two or more agents to dissociation after dilution.
  • the terms “immunoreactive” and “preferentially binds” are used interchangeably herein with respect to antibodies and/or antigen-binding fragments.
  • binding refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bond interactions, including interactions such as salt bridges and water bridges.
  • Non-specific binding would refer to binding with an affinity of less than about 10 7 M, e.g., binding with an affinity of 10 6 M, 10 5 M, 10 4 M, etc.
  • cell surface expression system or “cell surface display system” refers to the display or expression of a protein or portion thereof on the surface of a cell.
  • a cell is generated that expresses proteins of interest fused to a cell-surface protein.
  • a protein is expressed as a fusion protein with a transmembrane domain.
  • the term “element” includes polypeptides, including fusions of polypeptides, regions of polypeptides, and functional mutants or fragments thereof and polynucleotides, including microRNAs and shRNAs, and functional mutants or fragments thereof.
  • region is any segment of a polypeptide or polynucleotide.
  • a “domain” is a region of a polypeptide or polynucleotide with a functional and/or structural property.
  • the terms "stalk” or “stalk domain” refer to a flexible polypeptide connector region providing structural flexibility and spacing to flanking polypeptide regions and can consist of natural or synthetic polypeptides.
  • a stalk can be derived from a hinge or hinge region of an immunoglobulin (e.g., IgGl) that is generally defined as stretching from Glu216 to Pro230 of human IgGl (Burton (1985) Molec. Immunol., 22: 161-206).
  • Hinge regions of other IgG isotypes may be aligned with the IgGl sequence by placing the first and last cysteine residues forming inter-heavy chain disulfide (S-S) bonds in the same positions.
  • the stalk may be of natural occurrence or non-natural occurrence, including but not limited to an altered hinge region, as disclosed in U.S. Pat. No. 5,677,425.
  • the stalk can include a complete hinge region derived from an antibody of any class or subclass.
  • the stalk can also include regions derived from CD 8, CD28, or other receptors that provide a similar function in providing flexibility and spacing to flanking regions.
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
  • polypeptide is a single chain of amino acid residues linked by peptide bonds. A polypeptide does not fold into a fixed structure nor does it have any posttranslational modification. A “protein” is a polypeptide that folds into a fixed structure. “Polypeptides” and “proteins” are used interchangeably herein.
  • a polypeptide may be “purified” to remove contaminant components of a polypeptide’s natural environment, e.g., materials that would interfere with diagnostic or therapeutic uses for the polypeptide such as, for example, enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • a polypeptide can be purified (1) to greater than 90%, greater than 95%, or greater than 98%, by weight of antibody as determined by the Lowry method, for example, more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) under reducing or nonreducing conditions using Coomassie blue or silver stain.
  • SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis
  • the term “immune cells” generally includes white blood cells (leukocytes) which are derived from hematopoietic stem cells (HSC) produced in the bone marrow.
  • Immune cells includes, e.g., lymphocytes (T cells, B cells, natural killer (NK) cells) and myeloid-derived cells (neutrophil, eosinophil, basophil, monocyte, macrophage, dendritic cells).
  • T cell includes all types of immune cells expressing CD3 including T-helper cells (CD4 + cells), cytotoxic T cells (CD8 + cells), T-regulatory cells (Treg) and gamma-delta T cells.
  • NKT cells which are CD3+, CD56+, and either CD4+ or CD8+, are considered a type of T cells herein.
  • Surface expression of CD3 can be transiently decreased or eliminated in T cells, as has been observed with some of the methods for modifying T cells disclosed herein.
  • Such modified CD4+ or CD8+ lymphocytes that have transiently decreased/absent CD3 surface expression are still considered T cells in this disclosure.
  • Reference to a “CD” or cluster of differentiation marker, such as CD3+, CD4+, CD8+, CD56+ herein, relates to surface expression of such polypeptide.
  • surface expression is a continuum between positive and negative, and can be assessed by FACS analysis, where cells are determined to be positive or negative based on user cutoffs known in the art.
  • NK cell includes lymphocytes that express CD56 on their surface (CD56+ lymphocytes). NKT cells, which are CD3+, CD56+, and either CD4+ or CD8+, are considered a type of NK cells herein.
  • a "cytotoxic cell” includes CD8 + T cells, natural-killer (NK) cells, NK-T cells, y5 T cells, a subpopulation of CD4 + cells, and neutrophils, which are cells capable of mediating cytotoxicity responses.
  • stem cell generally includes pluripotent or multipotent stem cells.
  • stem cells includes, e.g., embryonic stem cells (ES); mesenchymal stem cells (MSC); induced- pluripotent stem cells (iPS); and committed progenitor cells (hematopoietic stem cells (HSC); bone marrow derived cells, etc.).
  • ES embryonic stem cells
  • MSC mesenchymal stem cells
  • iPS induced- pluripotent stem cells
  • HSC hematopoietic stem cells
  • bone marrow derived cells etc.
  • treatment refers to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, e.g., in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.
  • the terms “individual”, “subject”, “host”, and “patient” refer to a mammal, including, but not limited to, humans, murines (e.g., rats, mice), lagomorphs (e.g., rabbits), nonhuman primates, humans, canines, felines, ungulates (e.g., equines, bovines, ovines, porcines, caprines), etc.
  • the terms “therapeutically effective amount” or “efficacious amount” refers to the amount of an agent, or combined amounts of two agents, that, when administered to a mammal or other subject for treating a disease, is sufficient to affect such treatment for the disease. The “therapeutically effective amount” will vary depending on the agent(s), the disease and its severity and the age, weight, etc., of the subject to be treated.
  • the term “evolution” or “evolving” refers to using one or more methods of mutagenesis to generate a different polynucleotide encoding a different polypeptide, which is itself an improved biological molecule and/or contributes to the generation of another improved biological molecule.
  • Physiological or "normal” or “normal physiological” conditions are conditions such as, but not limited to, pressure, temperature, pH, ionic strength, osmotic pressure, osmolality, oxidative stress, concentration of one or more solutes, concentration of electrolytes, concentration of glucose, concentration of hyaluronan, concentration of lactic acid or lactate, concentration of albumin, levels of adenosine, levels of R-2-hydroxy glutarate, concentration of pyruvate, concentration of oxygen, and/or presence of oxidants, reductants, or co-factors, as well as other conditions, that 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.
  • a “transduced cell” or a “stably transfected cell” is a cell that contains an exogenous nucleic acid(s) that is integrated into the genome of the cell.
  • a “genetically modified cell” is a cell that contains an exogenous nucleic acid(s) regardless of whether the exogenous nucleic acid(s) is integrated into the genome of the cell, and regardless of the method used to introduce the exogenous nucleic acid(s) into the cell.
  • Exogenous nucleic acid(s) inside a cell that are not integrated into the genome of the cell can be referred to as “extrachromosomal” herein.
  • a “modified cell” is a cell that is associated with a recombinant nucleic acid vector (also called a “polynucleotide vector” or a “gene vector” herein), which in illustrative embodiments is a replication incompetent recombinant retroviral particle (also called a “RIR retroviral particle” or a “RIP” herein), that contains an exogenous nucleic acid, or a cell that has been genetically modified by an exogenous nucleic acid.
  • a recombinant nucleic acid vector also called a “polynucleotide vector” or a “gene vector” herein
  • a replication incompetent recombinant retroviral particle also called a “RIR retroviral particle” or a “RIP” herein
  • a modified cell associates with a replication incompetent recombinant retroviral particle through interactions between proteins on the surface of the cell and proteins on the surface of the replication incompetent recombinant retroviral particle, including pseudotyping elements and/or T cell activation elements.
  • a lipid-based reagent such as a liposomal reagent
  • the lipid-based reagent containing nucleic acid associates with the lipid bilayer of the modified cell before fusing or being internalized by the modified cell.
  • nucleic acid such as polyethylenimine (PEI) or calcium phosphate-based transfection
  • PKI polyethylenimine
  • the nucleic acid is typically associated with a positively charged transfection reagent to form the recombinant nucleic acid vector that associates with the negatively charged membrane of the modified cell before the complex is internalized by the modified cell.
  • Other means or methods of stably transfecting or genetically modifying cells include electroporation, ballistic delivery, and microinjection.
  • a “polypeptide” as used herein can include part of or an entire protein molecule as well as any posttranslational or other modifications.
  • a pseudotyping element as used herein can include a "binding polypeptide” that includes one or more polypeptides, typically glycoproteins, that identify and bind the target host cell, and one or more "fusogenic polypeptides” that mediate fusion of the retroviral and target host cell membranes, thereby allowing a retroviral genome to enter the target host cell.
  • the “binding polypeptide” as used herein can also be referred to as a “T cell and/or NK cell binding polypeptide” or a “target engagement element,” and the “fusogenic polypeptide” can also be referred to as a “fusogenic element”.
  • a “resting” lymphocyte such as for example, a resting T cell, is a lymphocyte in the GO stage of the cell cycle that does not express activation markers such as Ki-67. Resting lymphocytes can include naive T cells that have never encountered specific antigen and memory T cells that have been altered by a previous encounter with an antigen. A “resting” lymphocyte can also be referred to as a “quiescent” lymphocyte.
  • lymphodepletion involves methods that reduce the number of lymphocytes in a subject, for example by administration of a lymphodepletion agent. Lymphodepletion can also be attained by partial body or whole-body fractioned radiation therapy.
  • a lymphodepletion agent can be a chemical compound or composition capable of decreasing the number of functional lymphocytes in a mammal when administered to the mammal.
  • One example of such an agent is one or more chemotherapeutic agents.
  • Such agents and dosages are known, and can be selected by a treating physician depending on the subject to be treated.
  • lymphodepletion agents include, but are not limited to, fludarabine, cyclophosphamide, cladribine, denileukin diftitox, alemtuzumab or combinations thereof.
  • RNA interference is a biological process in which RNA molecules inhibit gene expression or translation by neutralizing targeted RNA molecules.
  • the RNA target may be mRNA, or it may be any other RNA susceptible to functional inhibition by RNAi.
  • an “inhibitory RNA molecule” refers to an RNA molecule whose presence within a cell results in RNAi and leads to reduced expression of a transcript to which the inhibitory RNA molecule is targeted.
  • An inhibitory RNA molecule as used herein has a 5’ stem and a 3’ stem that is capable of forming an RNA duplex.
  • the inhibitory RNA molecule can be, for example, a miRNA (either endogenous or artificial) or a shRNA, a precursor of a miRNA (i.e., a Pri-miRNA or Pre-miRNA) or shRNA, or a dsRNA that is either transcribed or introduced directly as an isolated nucleic acid, to a cell or subject.
  • a miRNA either endogenous or artificial
  • a shRNA a precursor of a miRNA (i.e., a Pri-miRNA or Pre-miRNA) or shRNA
  • a dsRNA that is either transcribed or introduced directly as an isolated nucleic acid, to a cell or subject.
  • double stranded RNA or “dsRNA” or “RNA duplex” refers to RNA molecules that are comprised of two strands. Double-stranded molecules include those comprised of two RNA strands that hybridize to form the duplex RNA structure or a single RNA strand that doubles back on itself to form a duplex structure. Most, but not necessarily all of the bases in the duplex regions are basepaired. The duplex region comprises a sequence complementary to a target RNA.
  • the sequence complementary to a target RNA is an antisense sequence, and is frequently from 18 to 29, from 19 to 29, from 19 to 21, or from 25 to 28 nucleotides long, or in some embodiments between 18, 19, 20, 21, 22, 23, 24, 25 on the low end and 21, 22, 23, 24, 25, 26, 27, 28 29, or 30 on the high end, where a given range always has a low end lower than a high end.
  • Such structures typically include a 5’ stem, a loop, and a 3’ stem connected by a loop which is contiguous with each stem and which is not part of the duplex.
  • the loop comprises, in certain embodiments, at least 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.
  • the loop comprises from 2 to 40, from 3 to 40, from 3 to 21, or from 19 to 21 nucleotides, or in some embodiments between 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 on the low end and 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40 on the high end, where a given range always has a low end lower than a high end.
  • microRNA flanking sequence refers to nucleotide sequences including microRNA processing elements.
  • MicroRNA processing elements are the minimal nucleic acid sequences which contribute to the production of mature microRNA from precursor microRNA. Often these elements are located within a 40-nucleotide sequence that flanks a microRNA stem-loop structure. In some instances, the microRNA processing elements are found within a stretch of nucleotide sequences of between 5 and 4,000 nucleotides in length that flank a microRNA stem-loop structure.
  • linker when used in reference to a multiplex inhibitory RNA molecule refers to a connecting means that joins two inhibitory RNA molecules.
  • a “recombinant retrovirus” refers to a non-replicable, or “replication incompetent”, retrovirus unless it is explicitly noted as a replicable retrovirus.
  • the terms “recombinant retrovirus” and “recombinant retroviral particle” are used interchangeably herein.
  • retrovirus/retroviral particle can be any type of retroviral particle including, for example, gamma retrovirus, and in illustrative embodiments, lentivirus.
  • retroviral particles typically are formed in packaging cells by transfecting the packing cells with plasmids that include packaging components such as Gag, Pol and Rev, an envelope or pseudotyping plasmid that encodes a pseudotyping element, and a transfer, genomic, or retroviral (e.g., lentiviral) expression vector, which is typically a plasmid on which a gene(s) or other coding sequence of interest is encoded.
  • packaging components such as Gag, Pol and Rev
  • an envelope or pseudotyping plasmid that encodes a pseudotyping element
  • a transfer, genomic, or retroviral (e.g., lentiviral) expression vector which is typically a plasmid on which a gene(s) or other coding sequence of interest is encoded.
  • a retroviral (e.g., lentiviral) expression vector includes sequences (e.g., a 5’ LTR and a 3’ LTR flanking e.g., a psi packaging element and a target heterologous coding sequence) that promote expression and packaging after transfection into a cell.
  • sequences e.g., a 5’ LTR and a 3’ LTR flanking e.g., a psi packaging element and a target heterologous coding sequence
  • lentivirus and “lentiviral particle” are used interchangeably herein.
  • a “framework” of a miRNA consists of “5’ microRNA flanking sequence” and/or “3’ microRNA flanking sequence” surrounding a miRNA and, in some cases, a loop sequence that separates the stems of a stem-loop structure in a miRNA.
  • the “framework” is derived from naturally occurring miRNAs, such as, for example, miR-155.
  • the terms “5’ microRNA flanking sequence” and “5’ arm” are used interchangeably herein.
  • the terms “3’ microRNA flanking sequence” and “3’ arm” are used interchangeably herein.
  • miRNA precursor refers to an RNA molecule of any length which can be enzymatically processed into an miRNA, such as a primary RNA transcript, a pri-miRNA, or a pre- miRNA.
  • construct refers to an isolated polypeptide or an isolated polynucleotide encoding a polypeptide.
  • a polynucleotide construct can encode a polypeptide, for example, a lymphoproliferative element.
  • a skilled artisan will understand whether a construct refers to an isolated polynucleotide or an isolated polypeptide depending on the context.
  • MOI Multiplicity of Infection ratio where the MOI is equal to the ratio of the number of virus particles used for infection per number of cells. Functional titering of the number of virus particles can be performed using FACS and reporter expression, as non-limiting examples.
  • PBMCs peripheral blood mononuclear cells
  • lymphocytes e.g., T cells, NK cells, and B cells
  • monocytes e.g., red blood cells, platelets and granulocytes (i.e., neutrophils, eosinophils, and basophils).
  • the term “or” is understood to be inclusive.
  • the term “and/or” as used in a phrase such as “A and/or B” herein includes each of the following: A and B; A or B; A (alone); and B (alone).
  • the term “and/or” as used in a phrase such as “A, B, and/or C” includes each of the following: A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone). This logic extends to any number of items in a list that are connected with the term “and/or”.
  • the present disclosure overcomes prior art challenges by providing improved methods and compositions for selectively killing or modulating the activity of modified cells, for example, modified NK cells and in illustrative embodiments, modified T cells, using anti-idiotype polypeptides expressed on the surface of the cells.
  • modified cells for example, modified NK cells and in illustrative embodiments, modified T cells
  • anti-idiotype polypeptides expressed on the surface of the cells.
  • anti-idiotype polypeptides that are improved cell therapy safety switches.
  • One challenge with prior art safety switches that are eTags is that they include EGFR family member domains, and thus are limited in structure to only EGFR domains. Furthermore, they may yield undesirable side effects when delivered to a subject since they are structurally related to a growth factor (e.g. EGFR) found in a subject.
  • a growth factor e.g. EGFR
  • anti-idiotype polypeptide safety switches are much more robust and flexible safety switches than eTags in terms of design and development.
  • Anti-idiotype polypeptide safety switch can be designed to be recognized by virtually any antibody, including any clinical antibody, such as a therapeutic antibody. Exemplary methods for developing antiidiotype antibodies, which utilize phage display assays, are disclosed in the Examples section herein.
  • the anti-idiotype polypeptides provided herein include designs that can be used to modulate cell activity, including, but not limited to, proliferation and apoptosis, and that do not require antibodies that drive cytotoxicity.
  • compositions herein provide versatile and effective solutions for controlling cytokine release syndrome in a subject with modified cells expressing a chimeric antigen receptor (CAR) or recombinant T cell receptor (TCR).
  • CAR chimeric antigen receptor
  • TCR recombinant T cell receptor
  • the methods provide an important step toward improving the safety and effectiveness of cell therapy methods.
  • Illustrative compositions for selectively killing of modified cells for example, modified NK cells and in illustrative embodiments, modified T cells, are easier to develop and safer than existing compositions and methods.
  • Some aspects of the anti-idiotype polypeptides provided herein, methods using the same, and polynucleotides encoding the same provide new methods and compositions to modulate the activity of the modified cells.
  • compositions that have many uses, including their use in these improved methods, are provided, including cell formulation compositions.
  • Some of these compositions include modified and in illustrative embodiments genetically modified lymphocytes that include safety switches for use with cellular therapies to affect the reduction or elimination of infused cells if dangerous adverse events develop.
  • Other of these compositions have improved proliferative and survival qualities, including in in vitro culturing, for example in the absence of growth factors.
  • modified and in illustrative embodiments genetically modified lymphocytes will have utility for example, as research tools to better understand mechanisms to control and stop cytokine release syndrome and the factors that influence T cell proliferation and survival, and for commercial production, for example for the production of certain factors, such as growth factors and immunomodulatory agents, that can be harvested and tested or used in commercial products. And such modified and genetically modified lymphocytes have utility in the treatment of cancer and other diseases.
  • anti-idiotype polypeptides and polynucleotides encoding these polypeptides (as disclosed in detail in other sections herein), that have numerous utilities in life sciences and medicine.
  • Such polypeptides in illustrative embodiments are especially useful in modified cells, for example for use in cell and gene therapy.
  • Anti-idiotype polypeptides expressed on the surface of cells can recognize target antibodies or target antibody mimetics that come in contact with these cells.
  • antibodies and antibody mimetics can be used to, for example mark cells expressing the anti-idiotype polypeptides for killing by the immune system, modulate a property (such as, for example, a proliferative state or an apoptotic state) or activity of the cells, label the cells, provide a target for enrichment and/or purification, enrich the cells, or cause the cells to aggregate.
  • a property such as, for example, a proliferative state or an apoptotic state
  • activity of the cells label the cells
  • provide a target for enrichment and/or purification enrich the cells, or cause the cells to aggregate.
  • a person skilled in the art will understand how to use the anti-idiotype polypeptides for these and other methods in view of the present disclosure. Accordingly, provided herein are methods for providing any of the above-mentioned uses, by expressing any of the polynucleotides that are disclosed herein, that include nucleic acids encoding anti-idiotype polypeptides disclosed herein.
  • FIG. 3 shows non-limiting exemplary embodiments of anti-idiotype polypeptides herein.
  • anti-idiotype polypeptides herein in illustrative aspects include an extracellular recognition domain (sometimes referred to as an anti-idiotype extracellular recognition domain, anti-id ERD, or anti-Id ECD and denoted in FIG. 3A-F and FIG. 3H as “anti-idiotype”) and typically include a membrane association domain (MAD), which is separated from the anti-id ECD in illustrative embodiments, by a stalk.
  • the anti-Id ERD in illustrative embodiments includes a recognition domain of an anti-idiotype antibody or anti-idiotype antibody mimetic.
  • an anti-idiotype polypeptide recognizes the idiotype of a target antibody or the idiotype of a target antibody mimetic.
  • an anti-idiotype extracellular recognition domain includes an idiotype-binding variable region of an anti-idiotype antibody or anti-idiotype antibody mimetic.
  • a stalk separates the MAD and the anti-idiotype extracellular recognition domain.
  • an extracellular recognition domain recognizes the idiotype of any antibody or antibody mimetic known in the art.
  • the extracellular recognition domain recognizes the idiotype of a clinical antibody or clinical antibody mimetic.
  • a clinical antibody in some illustrative embodiments, is a regulatory agency (e.g., U.S. FDA) approved biologic.
  • binding of the anti-idiotype polypeptide to the target antibody does not block or prevent binding between the target antibody and its cognate antigen.
  • binding of the anti-idiotype polypeptide to the target antibody blocks or prevents binding between the target antibody and its cognate antigen.
  • anti-idiotype polypeptides include a membrane association domain (sometimes referred to herein as MAD).
  • the membrane association domain of the anti-idiotype polypeptide attaches, tethers, or anchors the recognition domain from an anti-idiotype antibody or antibody mimetic to a cell membrane.
  • the membrane association domain comprises one or more of a transmembrane domain, and a GPI anchor, as further disclosed elsewhere herein.
  • the transmembrane domain can be a heterologous transmembrane domain, or an endogenous transmembrane domain, either of which could be the transmembrane domain of an antibody.
  • anti-idiotype polypeptides provided herein further include one or more intracellular domains (sometimes referred to herein as ICD).
  • the MAD of such anti-idiotype polypeptides that include ICDs in certain illustrative embodiments is a transmembrane domain (“TM” in FIGs. 3B-G).
  • the one or more intracellular domains can activate or inhibit pro-apoptotic or anti-apoptotic pathways and/or pro-survival or anti-survival pathways, and in certain embodiments modulate other cellular processes/pathways. Details are provided throughout this specification regarding these various embodiments and other embodiments wherein an anti-idiotype polypeptide herein, includes an ICD.
  • FIGs. 3B-G provides some non-limiting examples.
  • the ICD serves a structural role to assure the anti-id ERD is stably expressed on and remains bound to the cell membrane.
  • an ICD can have functional properties that are regulated by binding dimerization or multimerization that is induced by binding of the anti-id ERD by its target antibody or antibody mimetic.
  • the anti-id ERD (“anti-idiotype” in FIG. 3C) acts as an inducible extracellular dimerizing domain of an LE herein.
  • dimerization of an LE by binding of a target antibody to the anti-id ERD can activate signaling domains in the ICD, driving proliferation and/or cell survival.
  • the ICD of an LE can include P3 and optionally P4 domains, as disclosed herein with respect to LE ICDs.
  • the intracellular domains can activate one or more of a Jak/Stat pathway, a TRAF pathway, a PI3K pathway, or a PLC pathway.
  • lymphoproliferative section Disclosure related to mechanisms for activating these pathways is provided in the “Lymphoproliferative section” herein.
  • Illustrative examples of these embodiments are inducible chimeric lymphoproliferative elements, which are inducible upon binding of a target antibody to an extracellular domain that recognizes the idiotype of a target antibody.
  • the anti-id ERD (“anti-idiotype” in FIG. 3D) acts as the ASTR of a CAR or TCR.
  • the CAR or TCR includes the anti-id ERD, a stalk attaching the ERD to a transmembrane domain, and an ICD of a CAR or TCR.
  • binding of a target antibody to the anti-id ERD can activate signaling domains in the ICD of the CAR or TCR.
  • the intracellular domain is pro-apoptotic, and can include one or more intracellular signaling domains from a caspase protein and/or one or more intracellular signaling domains from tumor necrosis factor receptor superfamily members.
  • intracellular signaling domains from a caspase protein and/or one or more intracellular signaling domains from tumor necrosis factor receptor superfamily members.
  • such embodiments are specific examples of safety switches provided herein.
  • FIG. 3E such embodiments can include an anti-idiotype polypeptide wherein the ICD includes a death domain.
  • ICDs can include all or in certain illustrative embodiments, include a portion of an ICD from a TNF receptor superfamily member, that includes a death domain, such as FAS.
  • Illustrative embodiments of such an ant-idiotype polypeptide includes a constitutive dimerization domain in the extracellular domain, that can be all or part of the stalk or transmembrane domain, thus providing such anti-idiotype polypeptide, the ability to form higher order multimers, such as tetramers.
  • ICDs for any embodiment illustrated in FIG. 3E can include death domains, or other functional domains from initiator caspases, such as for example, caspases 2, 8, 9, and 10.
  • the anti-id ERD (“anti-idiotype” in FIG. 3D) acts as one of the ASTRs of a bi-specific CAR or TCR, wherein a second ASTR is present that typically binds to an antigen expressed by a cancer cell.
  • the CAR or TCR includes the anti-id ERD, a second ASTR, a stalk attaching the anti-id ERD and the 2 nd ASTR to a transmembrane domain, and an ICD of a CAR or TCR. Accordingly, binding of a target antibody to the anti-id ERD or binding of the 2 nd ASTR to its antigen, can activate signaling domains in the ICD of the CAR or TCR.
  • FIG. 3G illustrates another embodiment of anti-idiotype polypeptides herein that includes an ICD.
  • a cleavage site typically that is activated by dimerization, is added to the antiidiotype polypeptide between or as part of the transmembrane domain and the ICD.
  • these anti-idiotype polypeptides include a transmembrane domain that includes an amino acid sequence that serves as a substrate cleavage site upon dimerization, by certain proteases.
  • Such cleavage site can be, in certain embodiments, a substrate cleavage site for gamma-secretase complex, as discussed in more detail herein.
  • the intracellular domain for such embodiments can encode various intracellular polypeptides including certain caspases, including initiator caspases, for example caspases 2, 8, 9, and 10.
  • the ICD is a transcription factor.
  • the transcription factor is sequestered outside the nucleus until binding of the anti-id ERD by its target antibody inducing dimerization and cleavage, which releases the transcription factor such that it can enter the nucleus to become active in regulating gene expression.
  • FIG. 1 provides a non-limiting exemplary polynucleotide that is a portion of a lentiviral vector, in this non-limiting example a third-generation lentiviral expression vector, that includes nucleic acids that encode an anti-idiotype polypeptide.
  • the encoded anti-idiotype polypeptide includes an anti-idiotype extracellular recognition domain (“Anti-idiotype”), a stalk, and a membrane association domain (MAD).
  • Anti-idiotype anti-idiotype extracellular recognition domain
  • MAD membrane association domain
  • FIG. 1 includes a promoter (“Promoter”) positioned to drive expression of nucleic acids encoding different functional polypeptides including the anti-idiotype polypeptide. Furthermore, the polynucleotide vector of FIG. 1 includes various functional domains as indicated, including various viral functional domains, and encodes an optional inhibitory RNA molecule within the promoter.
  • Promoter a promoter positioned to drive expression of nucleic acids encoding different functional polypeptides including the anti-idiotype polypeptide.
  • the polynucleotide vector of FIG. 1 includes various functional domains as indicated, including various viral functional domains, and encodes an optional inhibitory RNA molecule within the promoter.
  • FIG. 2 provides non-limiting examples of some polynucleotides provided herein, that include nucleic acids that encode one or more anti-idiotype polypeptides, which themselves represent separate aspects herein.
  • nucleic acids that encode an anti-idiotype polypeptide in some embodiments, encode a membrane association domain (MAD) and an anti-idiotype extracellular recognition domain (referred to as “anti-idiotype” in FIG. 2).
  • nucleic acids encoding a stalk separate and are in frame with nucleic acids encoding the MAD and nucleic acids encoding the anti-idiotype extracellular recognition domain.
  • polynucleotides that encode additional functionalities expressed from the same promoter (i.e., on the same transcription unit) or from different promoters (i.e. different transcriptional units).
  • polynucleotides that include nucleic acids that encode an anti-idiotype extracellular recognition domain can include nucleic acids that encode, in addition to an anti-idiotype extracellular recognition domain, an engineered signaling polypeptide, a cytokine, and/or an inhibitory RNA.
  • a polynucleotide comprising: one or more transcriptional units, wherein each of the one or more transcriptional units is operatively linked to a promoter, wherein the one or more transcriptional units comprise: a) a polynucleotide sequence encoding one or more inhibitory RNA molecules, a first engineered signaling polypeptide, and/or a cytokine, and b) a polynucleotide sequence encoding an anti-idiotype polypeptide comprising an anti-idiotype extracellular recognition domain that recognizes an idiotype of a target antibody or a target antibody mimetic.
  • Polynucleotides that include nucleic acids that encode anti-idiotype polypeptides can be DNA or RNA. In some illustrative embodiments, they are mRNA. Such embodiments can include embodiments wherein the anti-idiotype antibody is directed against the antibody that forms the ASTR of a CAR. As such, mRNA that encode an anti-idiotype antibody can be directly delivered to a subject and when taken up and expressed by cells in the subject, such cells can form artificial antigen presenting cells that drive proliferation of CAR-T cells administered to the subject that express a CAR with an ASTR that is the target antibody recognized by the anti-idiotype polypeptide.
  • polynucleotides can be polynucleotide vectors, such as expression vectors. Further details regarding polynucleotides and polynucleotide vectors, such as RIPs, including lentiviral particles, are provided throughout this disclosure, including the claims. These further details include details regarding polynucleotides that include nucleic acids encoding anti-idiotype polypeptides. Some non-limiting noteworthy sections include the Recombinant Retroviral Particles section, the Nucleic Acids section, and the Exemplary Embodiments section.
  • Nucleic acids encoding the anti-idiotype polypeptide can be upstream or downstream (i.e. 5 ’or 3’) from those encoding other functionalities.
  • anti-idiotype polynucleotides are expressed as a separate polypeptide from other functional polypeptides.
  • polynucleotides herein encode an anti-idiotype polypeptide and are adapted for, structured for, and/or effective for expression in T cells and/or NK cells, and thus for T cell and/or NK cell therapies.
  • examples of such polynucleotides typically include a promoter that is active in T cells and/or NK cells, that drives expression of the anti-idiotype extracellular recognition domain and a membrane association domain, which thus are on the same transcriptional unit whose expression is driven by the promoter.
  • an anti-idiotype polypeptide is expressed as part of a single polynucleotide that also encodes a chimeric antigen receptor (CAR), an engineered T cell receptor (TCR) (FIG. 2B), a lymphoproliferative element (FIG. 2C), or a cytokine (FIG. 2D).
  • an anti-idiotype polypeptide is expressed as part of a single polynucleotide that encodes a CAR or a TCR, an anti-idiotype polypeptide, and a lymphoproliferative element (FIG. 2E), or a cytokine (FIG.
  • the polynucleotide encoding the anti-idiotype polypeptide is separated from the polynucleotide encoding the CAR, the TCR, the cytokine, and/or the polynucleotide encoding the lymphoproliferative element by an internal ribosome entry site (IRES) or a ribosomal skip sequence and/or cleavage signal (as shown, for example, in FIG. 2B-FIG. 2G).
  • IRES or ribosomal skip and/or cleavage signal can be any IRES or ribosomal skip sequence and/or cleavage signal known in the art.
  • the ribosomal skip sequence can be, for example, T2A with amino acid sequence GSGEGRGSLLTCGDVEENPGP (SEQ ID NO:83).
  • Other examples of cleavage signalsand/or ribosomal skip sequences include FMDV 2 A (F2A); equine rhinitis A virus 2 A (abbreviated as E2A); porcine teschovirus-1 2A (P2A); and Thoseaasigna virus 2A (T2A).
  • FIG. 4 provides additional examples of anti-idiotype polynucleotides provided herein.
  • FIG. 4A illustrates a polynucleotide construct that includes nucleic acids encoding a bispecific CAR (i.e. a CAR with an anti-id ERD as one ASTR), followed by a nucleic acid T2A element, followed by nucleic acids encoding an LE.
  • a bispecific CAR i.e. a CAR with an anti-id ERD as one ASTR
  • T2A element i.e. a nucleic acid with an anti-id ERD as one ASTR
  • FIG. 4B illustrates a polynucleotide construct that includes nucleic acids encoding a CAR or TCR, followed by a nucleic acid T2A element, followed by nucleic acids encoding an anti-idiotype polypeptide that includes an anti-id ERD, a stalk, a transmembrane domain, and an intracellular domain.
  • FIG. 4C illustrates a polynucleotide construct that includes nucleic acids encoding an anti-idiotype polypeptide that includes an anti-id ERD, a stalk, a transmembrane domain, and an intracellular domain, followed by a nucleic acid T2A element, followed by nucleic acids encoding an LE.
  • FIG. 4D illustrates a polynucleotide construct that includes nucleic acids encoding an anti-idiotype polypeptide that includes an anti-id ERD, a stalk, a transmembrane domain, and an intracellular domain, followed by a nucleic acid T2A element, followed by nucleic acids encoding a cytokine.
  • FIG. 4E illustrates a polynucleotide construct identical to FIG. 4B, but where the nucleic acids encoding the ICD of the anti-idiotype polypeptide are followed by a nucleic acid T2A element, which is followed by nucleic acids encoding an LE.
  • FIG. 4F illustrates a polynucleotide construct identical to FIG.
  • FIG. 4G illustrates a polynucleotide construct identical to FIG. 4E, but where the nucleic acids encoding the ICD of the anti-idiotype polypeptide are followed by a nucleic acid T2A element, which is followed by nucleic acids encoding a cytokine.
  • Fl-3-247 encoding a CD19 CAR and a polypeptide lymphoproliferative element comprised from amino to carboxy terminus of the Kozak-type sequence GCCGCCACCAT/UG(G) (SEQ ID NO:331), having the T at the “T/U” residue and having the optional last G, the CD8 signal peptide MALPVTALLLPLALLLHAARP (SEQ ID NO:72) (in which the sequence ATGG from the Kozak-type sequence also encodes the first four nucleotides of the CD8 signal peptide), a FLAG-TAG (DYKDDDDK; SEQ ID NO:74), a linker (GSTSGS; SEQ ID NO:349), an anti-CD19scFv, a CD8 stalk and transmembrane region, and an intracellular domain from CD3z followed by T2A and the lymphoproliferative element comprising the parts E006-T016-S186-S050 which encode an extracellular domain
  • F1-3-P100 encoding a CD19 CAR and an anti-idiotype polypeptide with the P3 and P4 domains of an LE comprised, from amino to carboxy terminus, of the Kozak-type sequence GCCGCCACCAT/UG(G) (SEQ ID NO:331), having the T at the “T/U” residue and having the optional last G, the CD8 signal peptide MALPVTALLLPLALLLHAARP (SEQ ID NO:72) (in which the sequence ATGG from the Kozak-type sequence also encodes the first four nucleotides of the CD8 signal peptide), a FLAG-TAG (DYKDDDDK; SEQ ID NO:74), a linker (GSTSGS; SEQ ID NO:349), an antiCD 19scFv, a CD8 stalk and transmembrane region, and an intracellular domain from CD3z followed by T2A an anti-cetuximab scFv from the table shown in FIG.
  • FIG. 5A shows a schematic of an illustrative bicistronic lentiviral genomic vector with divergent transcriptional units.
  • a first transcriptional unit comprising a lymphoproliferative element that is also anti-idiotype polynucleotide, followed by a poly adenylation sequence (Poly A) under the transcriptional control of an NFAT-responsive minimal IL-2 promoter (6x NF AT) is encoded in the reverse orientation.
  • an insulator element Ins separates the first and second transcriptional units.
  • the second transcriptional unit encodes a CAR (CAR) under the transcriptional control of a constitutive promoter (Promoter) and is encoded in the forward orientation.
  • CAR CAR
  • Triangles shown in dashed lines represent 3 possible locations into any one or more of which, one or more miRNAs could optionally be inserted into the vector.
  • the triangle shown in a dotted line represents 1 possible location in an exon within a promoter such as for EFl -a into which one or more inhibitory RNAs could optionally be inserted into the vector.
  • SA and SD correspond to splice donor and splice acceptor sites.
  • FIG. 5B shows a schematic of an illustrative bicistronic lentiviral genomic vector with divergent transcriptional units.
  • a first transcriptional encoding a lymphoproliferative element followed by a poly adenylation sequence (Poly A) under the transcriptional control of an NFAT-responsive minimal IL-2 promoter (6x NF AT) is encoded in the reverse orientation.
  • an insulator element (Ins) separates the first and second transcriptional units.
  • the second transcriptional unit encodes a CAR (CAR) followed by a T2A element and an anti-idiotype extracellular recognition domain, a stalk, and a MAD under the transcriptional control of a constitutive promoter (Promoter) and is encoded in the forward orientation.
  • CAR CAR
  • Promoter constitutive promoter
  • Triangles shown in dashed lines represent 3 possible locations into any one or more of which, one or more miRNAs could optionally be inserted into the vector.
  • the triangle shown in a dotted line represents 1 possible location in an exon within a promoter such as for EFl -a into which one or more inhibitory RNAs could optionally be inserted into the vector.
  • SA and “SD” correspond to splice donor and splice acceptor sites.
  • the ECD of the lymphoproliferative element in this design optionally comprises an eTAG or second anti-idiotype extracellular recognition domain.Fl -6-744 encodes a bicistronic lenti viral genomic vector with divergent transcriptional units.
  • the first transcriptional unit encodes the lymphoproliferative element E016-T016-S186-S050 under the control of an NFAT-responsive minimal IL-2 promoter all encoded in the reverse orientation.
  • the second transcriptional unit encodes a HER2 CAR comprised of an anti-HER2scFv, a CD8 stalk and transmembrane region, a CD 137 intracellular domain, and an intracellular activating domain from CD3z followed by T2A and an eTag.
  • the first and second transcriptional units are separated by the 250 cHS4 insulator (SEQ ID NO:358) in the forward orientation.
  • modified cells and in illustrative embodiments genetically modified cells that include any of the anti-idiotype polynucleotides provided herein.
  • modified cells, or in illustrative embodiments genetically modified cells are provided herein, that expresses any of the anti-idiotype polypeptides provided herein.
  • the modified or genetically modified cells can further express inhibitory RNA molecules and/or other polypeptides disclosed herein, for example, an engineered signaling polypeptide.
  • the modified or genetically modified cells can express an anti-idiotype polypeptide and a lymphoproliferative element, CAR, and/or recombinant TCR.
  • the modified or genetically modified cells can express an anti-idiotype polypeptide and a cytokine.
  • the cytokine is in a secreted form.
  • the cytokine is membrane-associated.
  • the cell is an immortalized cell.
  • the cells expressing an anti-idiotype polypeptide are lymphocytes, such as TILs, lymphocytes other than B cells, and in illustrative embodiments, T cells and/or NK cells.
  • the cells express a chimeric TCR or the cells are CAR-T cells and/or CAR-NK cells, or tumor infiltrating lymphocytes.
  • the T or NK cells are NKT cells. NKT cells are a subset of T cells that express CD3 and typically co-express an a[3 T-cell receptor, but also express a variety of molecular markers that are typically associated with NK cells (such as NK1.1 or CD56).
  • the cells are primary cells. In some embodiments, the cells are human cells. In some embodiments, the cells do not ordinarily produce antibodies. A skilled artisan will understand that reference to a lymphocyte that does not express an antibody in the context of a modified cell that includes or expresses an anti-idiotype polypeptide herein, refers to the native capability of the cell, and that such a modified cell includes, and in illustrative embodiments expresses, an anti-idiotype polypeptide.
  • the modified cells that have polynucleotides that include nucleic acids that encode an anti-idiotype polypeptide are present in a cell suspension within a commercial container, such as a commercial container suitable for cell therapy, such as a cell cryopreservation infusion bag.
  • a commercial container suitable for cell therapy such as a cell cryopreservation infusion bag.
  • the number of cells in the suspension within the commercial container can be sufficient to provide between 1 x 10 5 cells and 1 x 10 9 , between 1 x 10 6 cells and 1 x 10 9 , or between 1 x 10 6 cells and 5 x 10 8 , for example CAR-positive viable T cells and/or NK cells per kg of body weight of the subject to which the cells are to be delivered.
  • the commercial container can include the aforementioned ranges x 50-150 kg, or 50-100kg.
  • the commercial container includes between 1 x 10 7 and 1 x 10 11 cells, between 1 x 10 8 and 1 x 10 11 cells, or between 1 x 10 8 and 5 x 10 10 cells, modified cells, for example CAR-positive viable T cells and/or NK cells, and/or in an illustrative embodiment, cells that are positive for an anti-idiotype extracellular recognition domain.
  • anti-idiotype polypeptides herein can be used to confirm that modified cells that express a CAR, also express the anti-idiotype polypeptide, for example as a release criteria to help assure that the safety switch is present on such cells. Further details regarding modified cells herein, in commercial containers, can be found herein, for non-limiting example, within the “KITS AND COMMERCIAL PRODUCTS” section.
  • a modified or genetically modified T cell or NK cell made using a method according to any of the methods provided herein, wherein the cell is modified to contain a polynucleotide that includes nucleic acids that encode an anti-idiotype polypeptide.
  • the T cell or NK cell has been further modified to express a first engineered signaling polypeptide.
  • the first engineered signaling polypeptide can be an LE, a TCR, or a CAR that includes an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activating domain.
  • the T cell or NK cell can further include a second engineered signaling polypeptide that can be a CAR, a TCR, or a lymphoproliferative element.
  • the lymphoproliferative element can be a chimeric lymphoproliferative element.
  • the T cell or NK cell can further include a pseudotyping element on a surface.
  • the T cell or NK cell can further include an activation element on a surface.
  • the CAR, lymphoproliferative element, pseudotyping element, and activation element of the genetically modified T cell or NK cell can include any of the aspects, embodiments, or sub-embodiments disclosed herein.
  • the activation element can be anti-CD3 antibody, such as an anti-CD3 scFvFc or an anti-CD3 antibody mimetic.
  • the activation element can be anti-CD3 antibody, such as an anti-CD3 scFvFc or an anti-CD3 antibody mimetic.
  • TILs in illustrative embodiments TILs, T cells and/or NK cells, or self-driving CAR aspects provided herein, that relate to either aspects for transduction of T cells and/or NK cells in blood or a component thereof, that include transcription units that encode one, two, or more (e.g., 1-10, 2-10, 4-10, 1-6, 2-6, 3-6, 4-6, 1-4, 2-4, 3-4) inhibitory RNA molecules.
  • inhibitory RNA molecules are lymphoproliferative elements and therefore, can be included in any aspect or embodiment disclosed herein as the lymphoproliferative element as long as they induce proliferation of a T cell and/or an NK cell, or otherwise meet a test for a lymphoproliferative element provided herein.
  • the ASTR of the CAR is an MRB ASTR and/or the ASTR of the CAR binds to a tumor associated antigen.
  • the first nucleic acid sequence is operably linked to a riboswitch, which for example is capable of binding a nucleoside analog, and in illustrative embodiments is an antiviral drug such as acyclovir.
  • control element is a polynucleotide comprising a riboswitch.
  • the riboswitch is capable of binding a nucleoside analog and when the nucleoside analog is present, one or both of the engineered signaling polypeptides are expressed.
  • a cell of the current disclosure for example a lymphocyte, such as a primary cell, and/or a lymphocyte that does not naturally produce antibodies, and in illustrative embodiments, a T cell and/or NK cell, can include more than one type of anti-idiotype polypeptide on its surface, i.e., anti-idiotype polypeptides with different extracellular recognition domains.
  • a cell can include a first anti-idiotype polypeptide on its surface that recognizes a first target antibody or antibody mimetic and elicits a first response upon binding, and a second anti-idiotype polypeptide on its surface that recognizes a second target antibody or antibody mimetic and elicits a second response upon binding.
  • a polynucleotide encodes one, two, three, or four, or more anti-idiotype polypeptides.
  • a cell contains such a polynucleotide, and expresses the one, two, three, or four, or more encoded anti-idiotype polypeptides.
  • Safety switches have been developed for use with cellular therapies to affect the reduction or elimination of infused cells in the case of adverse events.
  • Any of the replication incompetent recombinant retroviral particles provided herein can include nucleic acids that encode a safety switch as part of, or separate from, nucleic acids encoding any of the engineered signaling polypeptides provided herein.
  • any of the engineered signaling polypeptides provided herein for example engineered signaling polypeptides in modified, genetically modified, and/or transduced lymphocytes to be introduced or reintroduced into a subject, can include a safety switch.
  • any of the engineered T cells disclosed herein can include a safety switch.
  • Safety switch technologies can be broadly categorized into three groups based on their mechanism of action; antibody- or antibody mimetic-mediated cytotoxicity, pro-apoptotic signaling, and metabolic (gene-directed enzyme prodrug therapy, GDEPT).
  • Previously disclosed safety switches include cell surface molecules that are truncated tyrosine kinase receptors.
  • the truncated tyrosine kinase receptor is a member of the epidermal growth factor receptor (EGFR) family (e.g., ErbBl (HER1), ErbB2, ErbB3, and ErbB4), for example as disclosed in U.S. Patent 8,802,374 or
  • eTags are recognized by monoclonal antibodies that are commercially available such as matuzumab, necitumumab panitumumab, and in illustrative embodiments, cetuximab.
  • eTag was demonstrated to have a cell killing potential through Erbitux® mediated antibody dependent cellular cytotoxicity (ADCC) pathways.
  • ADCC antibody dependent cellular cytotoxicity
  • the inventors of the present disclosure have successfully expressed eTag in PBMCs using lentiviral vectors, and have found that expression of eTag in vitro by PBMCs exposed to Cetuximab, provided an effective elimination mechanism for PBMCs.
  • eTags can be used in some embodiments of the present disclosure, but in such embodiments, typically an anti-idiotype extracellular domain is present as well.
  • the extracellular recognition domain i.e. cell tag
  • the extracellular recognition domain is itself an antibody, which as disclosed herein includes a functional antibody fragment, that binds a predetermined binding partner antibody (e.g. a target antibody).
  • the cell tag antibody is specific for the target antibody, and for example, does not bind antibody constant regions exclusively, or in some embodiments, at all, or in illustrative embodiments, unless they interact with the target antibody (Abl) to cell tag (extracellular recognition domain) (Ab2) binding.
  • the cell tag antibody i.e. extracellular recognition domain that includes the variable region of an antibody
  • the anti-idiotypic antibody recognizes an epitope on the predetermined binding partner antibody (i.e. target antibody) (Abl) that is distinct from the antigen binding site on Abl.
  • Ab2 binds the variable region of Abl.
  • Ab2 binds the antigen-binding site of Abl, and in illustrative embodiments, competes with Abl for binding to the antigen-binding site of Abl.
  • Ab2 may be from any animal including human and murine, or humanized or a chimeric antibody or an antibody derivative included within the definition of “antibody” herein, including, for example antibody fragments (Fab, Fab’, F(ab’)2, scFv, diabodies, bispecific antibodies, and antibody fusion proteins.
  • Ab2 is typically associated with a membrane through a membrane association domain.
  • Ab2 is associated with the cell surface via its endogenous transmembrane domain.
  • Ab2 is associated with the cell surface via a heterologous transmembrane domain or membrane attachment sequence such as GPI.
  • Abl is a commercially available monoclonal antibody.
  • Abl is a commercially available monoclonal antibody therapeutic. In further illustrative embodiments, Abl is capable of mediating ADCC and/or CDC as described below.
  • safety switches can also function as flags that label or mark polynucleotides, polypeptides, or cells as being engineered.
  • Such safety switches can be detected using standard laboratory techniques including PCR, Southern Blots, RT-PCR, Northern Blots, Western Blots, histology, and flow cytometry.
  • detection of eTAG by flow cytometry has been used by at least one of the inventors as an in vivo backing marker for T cell engraftment in mice.
  • cell tags are used to enrich for engineered cells using antibodies or ligands optionally bound to a solid substrate such as a column or beads.
  • biotinylated-cetuximab to immunomagnetic selection in combination with anti-biotin microbeads successfully enriches T cells that have been lentivirally transduced with eTAG containing constructs from as low as 2% of the population to greater than 90% purity without observable toxicity to the cell preparation.
  • the anti-idiotype polypeptide is a safety switch (also called a safety switch polypeptide or an anti-idiotype polypeptide safety switch herein) comprising a recognition domain of an anti-idiotype antibody or anti-idiotype antibody mimetic and a membrane association domain.
  • a safety switch polypeptide or an anti-idiotype polypeptide safety switch herein
  • Such safety switch polypeptides can be designed much more efficiently and with many more optional sequences and designs, than prior art safety switches.
  • Such a safety switch polypeptide in one aspect is designed such that the extracellular recognition domain recognizes an idiotype of an antibody or antibody mimetic capable of inducing cytotoxicity.
  • the safety switch is based on antibody mediated cytotoxicity upon antibody or antibody mimetic binding to an anti-idiotype polypeptide expressed on the surface of a cell, and more specifically binding to an extracellular recognition domain (also referred to herein as a cell tag or more specifically, an anti-idiotype cell tag) of an anti-idiotype polypeptide.
  • the antibody or antibody mimetic binds to the cell tag and induces complement-dependent cytotoxicity (CDC) and/or antibody-dependent cell-mediated cytotoxicity (ADCC).
  • binding of the antibody or antibody mimetic to the anti-idiotype polypeptide induces, promotes, and/or activates one or more of ADCC, CDC, antibody-mediated complement activation, antibody-dependent cellular phagocytosis, and antibody-dependent enhancement of diseases. Details related to other antibody and antibody mimetic functions, including corresponding Fc domains for eliciting such responses, are discussed in the “Antibody and antibody mimetic effector functions” herein.
  • the anti-idiotype polypeptide can be immunogenic, to further stimulate the immune system.
  • the cell tag is immunogenic.
  • the cell tag polypeptide is non-immunogenic.
  • a safety switch polypeptide is designed such that the anti-idiotype polypeptide includes an intracellular domain having one or more cell-death inducing signals, and the polypeptide is capable of inducing a cell death signal upon binding of the anti-idiotype polypeptide to a target antibody or antibody mimetic that comprises the idiotype recognized by the anti-idiotype polypeptide.
  • the cell-death inducing signals can be induced based on dimerization-induced apoptotic signaling.
  • the safety switch is based on dimerization induced apoptotic signals.
  • such a safety switch comprises an extracellular dimerization domain comprising a recognition domain of an anti-idiotype antibody or antibody mimetic linked in frame with a membrane association domain and an intracellular domain comprising components of an apoptotic pathway.
  • dimerization mediated by the binding of an antibody or antibody mimetic to the anti-idiotype polypeptide results in apoptosis of the cell.
  • the safety switch includes inducible FAS (iFAS) comprised of one or more inducible dimerization domains, i.e., the anti-idiotype polypeptides, fused to the cytoplasmic tail of the Fas receptor and localized to the membrane by a membrane association domain.
  • the safety switch includes one or more domains from a Caspase, such as caspase- 1 or caspase-9.
  • the anti-idiotype polypeptides can be expressed as fusions with other polypeptides disclosed herein, including a lymphoproliferative element, a CAR, and/or a recombinant TCR. In other embodiments, the anti-idiotype polypeptides are expressed as polypeptides by themselves.
  • the anti-idiotype polypeptides can include any of the domains disclosed herein to be included in a lymphoproliferative element, CAR, and/or TCR, such as the extracellular domains, stalks, transmembrane domains, intracellular activating domains, modulatory domains, linkers, or intracellular domains.
  • the anti-idiotype compositions provided herein have many uses, including in in vitro and in vivo methods for cell tagging for example, and in illustrative embodiments, in methods of delivering polynucleotides or polynucleotide vectors (e.g., RIPs) and/or modified cells to a subject.
  • Such methods for delivering polynucleotides, polynucleotide vectors and/or modified cells to a subject can for example, utilize signaling that is regulated by binding of a target antibody to an anti-idiotype extracellular recognition domain of an anti-idiotype polypeptide provided herein.
  • Such a method can be a method for inducing target cell proliferation or death, wherein the target cells are the modified cells that are delivered, or are cells present in the subject that are modified in vivo in the subject by transduction, transfection, electroporation, or other means of gene delivery, with polynucleotides or polynucleotide vectors encoding anti-idiotype polynucleotides that are directly delivered in some embodiments.
  • Target cell proliferation or death in illustrative embodiments is induced by binding of a target antibody to an anti-idiotype extracellular recognition domain expressed on the target cell.
  • a method for administering modified cells and/or polynucleotides, such as polynucleotide vectors, that include nucleic acids that encode an anti-idiotype polypeptide to a mammalian subject includes delivering the polynucleotides, such as polynucleotide vectors and/or modified cells to the mammalian subject.
  • the polynucleotides or polynucleotide vectors can be any of the polynucleotide vectors provided herein that comprise nucleic acids encoding an anti-idiotype polypeptide provided herein.
  • anti-idiotype polypeptides in certain embodiments include a membrane association domain, which in illustrative embodiments is separated from the anti-idiotype extracellular recognition domain on the anti-idiotype polypeptide, by a stalk.
  • the anti-idiotype polypeptide includes an intracellular domain, which in some embodiments is an intracellular signaling domain.
  • the modified cells can be any of the modified cells provided herein that comprise, and in illustrative embodiments express, any of the polynucleotides provided herein that comprise nucleic acids that encode an anti-idiotype polypeptide.
  • the modified cells are genetically modified cells.
  • Such modified cells, or genetically modified cells in some non-limiting embodiments are lymphocytes or lymphocytes other than B cells, and in some embodiments T cells and/or NK cells, such as CAR-T cells and/or CAR- NK cells.
  • the polynucleotides are RNA or DNA, and in illustrative embodiments are mRNA, for example a synthetic anti-idiotype mRNA as disclosed herein.
  • the polynucleotide vectors are replication incompetent retroviral particles (RIPs), for example recombinant lenti viral particles.
  • RIPs replication incompetent retroviral particles
  • Systems and methods for processing cells that are removed from the subject can include traditional closed cell processing systems and methods, or “more recent” methods and systems as disclosed in further detail herein.
  • PBMCs peripheral blood mononuclear cells
  • lymphocytes typically T cells and/or NK cells
  • PBMCs peripheral blood mononuclear cells
  • resting T cells and/or resting NK cells in illustrative embodiments with polynucleotides that include nucleic acids encoding anti-idiotype polypeptides, in a reaction mixture.
  • Such methods can include a contacting step that includes contacting lymphocytes with polynucleotide vectors, such as, but not limited to, replication incompetent recombinant retroviral particles (RIPs) in the reaction mixture, that include nucleic acids that encode an anti-idiotype polypeptide.
  • polynucleotide vectors such as, but not limited to, replication incompetent recombinant retroviral particles (RIPs) in the reaction mixture, that include nucleic acids that encode an anti-idiotype polypeptide.
  • RIPs replication incompetent recombinant retroviral particles
  • Such reaction mixture themselves represent separate aspects provided herein.
  • the reaction mixture comprises blood, or a component thereof, and/or an anticoagulant.
  • the reaction mixture in illustrative embodiments comprises lymphocytes and identical copies of polynucleotide vectors, such as a replication incompetent recombinant retroviral particles, that comprise nucleic acids that encode an anti-idiotype polypeptide
  • the reaction mixture can include a T cell activation element.
  • the reaction mixture includes one or more additional blood components set out below that in illustrative embodiments are present because the reaction mixture comprises at least 10% whole blood.
  • the RIPs comprise a T cell binding polypeptide and a fusogenic polypeptide on their surface, which in illustrative embodiments is a pseudotyping element.
  • the contacting and incubation under contacting conditions) facilitates association of the lymphocytes with the RIPs, wherein the RIPs genetically modify and/or transduce the lymphocytes.
  • the RIPs can further include a T cell activation element on their surface.
  • the methods further include instructing a medical professional, patient or caregiver that a target antibody should be administered to the subject if life-threatening adverse events develop, and/or delivering a target antibody or antibody mimetic to the subject in response to lifethreatening adverse events.
  • adverse events include grade 3 and/or grade 4 adverse events, including grade 3 and/or grade 4 CRS or ICANS (Lee et al, “ASTCT Consensus Grading for Cytokine Release Syndrome and Neurologic Toxicity Associated with Immune Effector Cells”; Biology of Blood and Marrow Transplantation; 25: 6235-638 (2019)).
  • the target antibody or antibody mimetic in illustrative embodiments, comprises an idiotype recognized by the anti-idiotype extracellular recognition domain.
  • the target antibody or antibody mimetic can be any of the target antibodies and antibody mimetics disclosed herein, including, but not limited to, approved biologic target antibodies and antibody mimetics.
  • the target antibody or antibody mimetic in some embodiments, is delivered in sufficient quantity to selectively kill at least 1%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 99% of the genetically modified T cells and/or NK cells in the subject.
  • Methods for delivering modified cells, polynucleotides, polynucleotide vectors, and antibody or antibody mimetics to a subject are known in the art.
  • delivery of the modified or genetically modified cells, polynucleotides or the polynucleotide vectors (e.g., RIPs), or the target antibody or antibody mimetic is performed by intravenous administration/infusion, intraperitoneal administration/injection, subcutaneous administration/injection, or intramuscular administration/injection.
  • the modified cells (e.g., lymphocytes) introduced into the subject are autologous cells, and in other embodiments they are allogeneic cells.
  • the cells typically are from a different person, and the cells (e.g., lymphocytes) from the subject are not modified. In some embodiments, no blood is collected from the subject to harvest cells during the method.
  • the present disclosure provides various treatment methods that involve delivery of an engineered T cell receptor or a CAR.
  • An engineered T cell receptor or a CAR of the present disclosure when present in a modified T lymphocyte or NK cell, or in a polynucleotide vector to be delivered directly to a subject, can mediate cytotoxicity toward a target cell.
  • Such methods typically involve administration of modified lymphocytes, or substantially purified or purified polynucleotide vectors (e.g., RIRs) to a subject as provided herein.
  • modified T cells and/or NK cells, polynucleotides, or polynucleotide vectors that are delivered into a subject are further modified such that they are capable of expressing, or express an anti-idiotype polypeptide and a CAR, a TCR, a cytokine, and/or a lymphoproliferative element as provided herein.
  • the modified cells express the anti-idiotype polypeptides and one or more of the CAR, the TCR, the cytokine, and/or the lymphoproliferative element from the same polynucleotide and sometimes on the same polypeptide, as disclosed herein.
  • the modified cells are lymphocytes, in illustrative embodiments T cells and/or NK cells.
  • such methods herein include delivery directly into a subject, of polynucleotides and polynucleotide vectors provided herein that include a promoter that is active in lymphocytes, in illustrative embodiments active in T cells and/or NK cells, and encode an anti-idiotype polypeptide.
  • Such methods that involve delivery of modified T cells and/or NK cells, or polynucleotide or polynucleotide vectors with promoters that are active in T cells and/or NK cells, are especially suited for, adapted for, and/or effective for methods in which the anti-idiotype polypeptides regulate T cell and/or NK cell proliferation (e.g., are useful as lymphoproliferative elements) and/or inducible cell killing (e.g., are useful as safety switches).
  • Methods herein where anti-idiotype polypeptides are used to promote selective killing of modified cells for example modified CAR-T, CAR-NK, or TIL cells, can be referred to herein as safety switch methods.
  • such safety switch methods utilize any of the polynucleotides that include nucleic acids encoding anti-idiotype polynucleotides, and corresponding encoded and in illustrative embodiments, expressed, anti-idiotype polypeptides, provided herein.
  • such safety switch methods involve delivery of modified CAR-T cells, modified CAR-NK cells, or modified TIL cells to the subject, and can involve delivery of a target clinical antibody.
  • Antibody delivery can be directed and administered to induce proliferation of the modified cells in the subject, and/or if the subject develops clinical symptoms that indicate that the modified cells that were delivered or cells derived therefrom, are causing adverse events in the subject, for example, grade 1 or grade 2 adverse events, and in some embodiments grade 3 and grade 4 adverse events, or otherwise putting the subject at risk of medical complications or even death.
  • a method, system, and kits wherein the same anti-idiotype polypeptide, or polynucleotide, which typically for these embodiments includes an intracellular proliferation-inducing signaling domain, can be used to induce proliferation or killing, depending on whether the antibody that contacts a modified cell expressing the anti-idiotype polypeptide, has a structure to induce cell killing or only dimerization of anti-idiotype polypeptides.
  • delivery of the target clinical antibody would be at a later date that is days, weeks, or months after the first or most recent delivery of the modified cells to the subject.
  • methods for administering a modified cell can include receiving information regarding adverse events of a subject and/or testing a subject for adverse events, and instructing a medical professional, a patient, and/or a caregiver to administer a target antibody if a patient develops certain adverse events, and in some embodiments such target antibody is administered.
  • the instructions can specify to administer the target antibody if the patient develops certain grade 3 or grade 4 adverse events.
  • adverse events can include, but are not limited to tumor lysis syndrome, cytokine release syndrome (CRS), macrophage activation syndrome, and/or neurotoxicity.
  • IFN interferon
  • MCP monocyte chemotactic protein
  • MIP macrophage inflammatory protein
  • some embodiments herein include detecting and/or receiving a report of decreased level of consciousness, confusion, seizures and cerebral edema.
  • a method of treating a disease or disorder in a subject having the disease or disorder can include, contacting a polynucleotide vector, such as an expression vector, including nucleic acid sequences encoding an anti-idiotype polypeptide provided herein, with peripheral blood cells obtained from the subject to produce modified T cells and/or NK cells, such as modified cytotoxic T cells and/or NK cells, before administering the modified T cells and/or NK cells to the subject.
  • a polynucleotide vector such as an expression vector, including nucleic acid sequences encoding an anti-idiotype polypeptide provided herein
  • methods provided herein such as methods for delivering modified cells to a subject, which can be adoptive cell therapies, methods for producing persistent populations of cells, methods for delivering a formulation, etc. as non-limiting examples, are especially adopted for treating cancer.
  • Such cancer can be any type of cancer.
  • such methods can be used for treating a subject who has, or a tumor associated with ovarian cancer, soft tissue sarcoma, peripheral T cell cancer, colorectal cancer, intrahepatic cholangiocarcinoma, glioblastoma, esophageal cancer, cutaneous T cell lymphoma, non-hodgkin lymphoma, urothelial cancer, basal cell carcinoma, epithelioid sarcoma, pancreatic cancer, non-small cell lung carcinoma, hodgkin lymphoma, renal cell carcinoma, mesothelioma, metastatic uveal melanoma, kidney cancer, blood cancer, HER2-expressing cancers, nonmelanoma skin cancer, liposarcoma, hepatocellular carcinoma, small lymphocytic lymphoma, prostate cancer, breast cancer, anal cancer, marginal zone lymphoma, cutaneous squamous cell carcinoma, thyroid cancer, medullary thyroid cancer, triple-negative breast cancer,
  • methods herein can be used to treat tumors that express any one or more of the tumor-associated antigens and/or tumor-specific antigens provided herein, and engineered T cell receptors and CARs can be designed to recognize such targets.
  • tumor associated or tumor specific antigens include blood tumor antigens provided herein elsewhere in this specification, and in some non-limiting embodiments includes the following antigens, most or all of which are believed to be associated with solid tumors: AXL, CD44v6, CAIX, CEA, CD133, c-Met, EGFR, EGFRvIII, Epcam, EphA2, GD2, GPC3, GUCY2C, HER1, HER2, ICAM-1, IL13Ra2, ILllRa, Kras, Kras G12D, L1CAM, MAGE, MET, Mesothelin, MUC1, MUC16 ecto, NKG2D, NY-ESO-1, PSCA, ROR-2, WT-1.
  • any of the methods provided herein that involve an administering step can be combined with administration of another cancer therapy, which in certain embodiments, can be a cancer vaccine, for example delivered subcutaneously.
  • another cancer therapy which in certain embodiments, can be a cancer vaccine, for example delivered subcutaneously.
  • such methods provided herein that include administering genetically modified T cells and/or NK cells into a subject, especially where the subject has, is afflicted with, or is suspected of having cancer can further include delivering an effective dose of an immune checkpoint inhibitor to the subject.
  • This checkpoint inhibitor delivery can occur before, after, or at the same time as administering the genetically modified T cells and/or NK cells.
  • Immune checkpoint inhibitors are known and various compounds are approved and in clinical development. Check point molecules, many of which are the target of checkpoint inhibitor compounds, include, but not limited to an anti-PDl antibody.
  • the administering is for treating cancer in the subject, and wherein a tumor in the subject regresses within 60 days, 45 days, 30 days, or 14 days after said administering.
  • the tumor is a blood cancer, for example DLBCL, that in illustrative examples expresses any of the blood cancer antigens provided herein.
  • the tumor is a solid tumor that expresses a solid tumor antigen, which in certain illustrative embodiments is a HER2 positive solid tumor, such as, but not limited to, breast cancer.
  • the administering is for treating cancer in the subject, and wherein the subject experiences stable disease, at least a partial response, or a complete response, in illustrative embodiments by RECIST1.1 criteria, within 90 days, 75 days, 60 days, 45 days, 30 days, or 14 days after said administering.
  • the tumor shrinks by at least 10%, 20%, 25%, 30%, 50% or more.
  • a partial response occurs when the sum of tumor lesions reduces by 30% or more and is confirmed at least 4 weeks after the prior scan without the appearance of new lesions and/or any pathological lymph nodes have a reduction in short axis to less than 10 mm.
  • a complete response occurs when all target and non-target lesions disappear.
  • the administering is for treating cancer in the subject, and wherein the subject experiences at least a partial response or experiences a complete response within 60 days, 45 days, 30 days, or 14 days after said administering.
  • the subject is a human afflicted with cancer.
  • the cell formulation is administered 2, 3, 4, 5, 6, or more times, or in illustrative embodiments only once to the subject before stable disease, or in illustrative embodiments a partial response or a complete response is achieved.
  • a second formulation is administered to the subject at a second, third, fourth, etc. timepoint between 1 day and 1 month, 2 months, 3 months, 6 months, or 12 months after the administering a first cell formulation, wherein the second formulation can be identical to the first formulation, or can comprises any of the formulations provided herein.
  • the method can be performed on a mammalian subject that has been subjected to a lymphodepletion process, as are known in the art.
  • the administration of the modified T cells and/or NK cells, or RER retroviral particles (RIPs) is performed in a method that does not require lymphodepletion of the subject for successful engraftment in the subject and/or for successful reduction of tumor volume in the subject, or that is performed on a mammalian (e.g.
  • the administration is performed on a mammalian (e.g. human) subject that is not suffering from a low white blood cell count, lymphopenia or lymphocytopenia.
  • the subcutaneous administration is performed on a subject having a lymphocyte count in the normal range (i.e., 1,000 and 4,800 lymphocytes in 1 microliter (pL) of blood).
  • the subcutaneous administration is performed on a subject having between 1,000 and 5,000, over 300, over 500, over 1,000, over 1,500, or over 2,000 lymphocytes per pL of blood).
  • the subcutaneous administration is performed on a mammalian (e.g., human) subject that is lymphoreplete.
  • Anti-idiotype polypeptides of the current disclosure bind to the idiotype of target antibodies and/or target antibody mimetics.
  • the target antibody can include one or more domains from or derived from a a mouse antibody, a rat antibody, a rabbit antibody, a goat antibody, a chicken antibody, a sheep antibody, a cow antibody, a llama antibody, a chimeric antibody, or in illustrative embodiments, a human antibody.
  • different target antibodies or target antibody mimetics can be used to bind to the same anti-idiotype polypeptide to elicit different responses, for example a first target antibody to promote cell proliferation and/or survival and a second target antibody mimetic to promote cell death.
  • the target antibody or antibody mimetic is a therapeutic antibody or a therapeutic antibody mimetic, respectively.
  • the target antibody or antibody mimetic can be a clinical antibody or clinical antibody mimetic, respectively.
  • the clinical antibody or clinical antibody mimetic is the subject of an FDA-approved Investigational New Drug Application (IND), or equivalent approved regulatory filing for initial clinical testing in humans in another country or jurisdiction.
  • IND Investigational New Drug Application
  • the target antibody or the target antibody mimetic as per the Investigational New Drug Application or equivalent is a stand-alone product, with no other active therapeutic or ingredient being tested as part of the IND.
  • the clinical antibody or antibody mimetic is a regulatory agency (e.g., U.S.
  • the target antibody can be one or more of cetuximab, muromonab-CD3, efalizumab, tositumomab-il31, nebacumab, edrecolomab, catumaxomab, daclizumab, olaratumab, abciximab, rituximab, basiliximab, palivizumab, infliximab, trastuzumab, adalimumab, ibritumomab tiuxetan, omalizumab, bevacizumab, natalizumab, panitumumab, ranibizumab, eculizumab, certolizumab pegol, ustekinumab, canakinumab, golimumab, ofatumumab, tocilizumab, denosumab, belimumab, i
  • binding of the anti-idiotype polypeptide to its target antibody or antibody mimetic prevents and/or blocks binding of the target antibody or antibody mimetic to the cognate antigen of the target antibody or antibody mimetic.
  • the extracellular recognition domain of the anti-idiotype polypeptide is capable of, adapted for, and/or configured to prevent and/or block binding of the target antibody or antibody mimetic to the cognate antigen of the target antibody or antibody mimetic when the anti-idiotype polypeptide is bound to the target antibody or antibody mimetic.
  • the target antibody is cetuximab.
  • binding of the anti-idiotype polypeptide to cetuximab prevents and/or blocks binding of cetuximab to Epidermal Growth Factor Receptor (EGFR).
  • EGFR Epidermal Growth Factor Receptor
  • the extracellular recognition domain of the antiidiotype polypeptide is capable of, adapted for, and/or configured to prevent and/or block binding of cetuximab to EGFR when the anti-idiotype polypeptide is bound to cetuximab.
  • the clinical antibody or clinical antibody mimetic has been shown in one or more clinical trials to have an acceptable safety (i.e., adverse event) profile.
  • the clinical antibody or clinical antibody mimetic has passed human clinical safety testing in a stand-alone clinical trial of the clinical antibody or clinical antibody mimetic.
  • the clinical antibody or clinical antibody mimetic for which a stand-alone application for regulatory approval has been filed with the Food and Drug Administration of the U.S. (USFDA), European Medicines Agency (EMA), National Medical Products Administration of China (NMPA) (Chinese FDA), or the Pharmaceutical and Food Safety Bureau (PFSB) of Japan.
  • the clinical antibody or clinical antibody mimetic for which an application for approval has been filed with the Food and Drug Administration of the U.S. (USFDA), European Medicines Agency (EMA), National Medical Products Administration of China (NMPA) (Chinese FDA), or the Pharmaceutical and Food Safety Bureau (PFSB) of Japan.
  • BLA Biologic Eicense Application
  • the clinical antibody or clinical antibody mimetic is an approved biologic antibody or antibody mimetic, approved by the Food and Drug Administration of the U.S. (USFDA), European Medicines Agency (EMA), National Medical Products Administration of China (NMPA) (Chinese FDA), or the Pharmaceutical and Food Safety Bureau (PFSB) of Japan.
  • the target antibody or antibody mimetic can be a bispecific antibody. In some embodiments, the target antibody or antibody mimetic can be a multispecific antibody. Multispecific antibodies have binding specificities for at least two different sites. In some embodiments, one of the binding specificities is for one target antigen and the other is for another target antigen. In some embodiments, bispecific antibodies or antibody mimetics may bind to two different epitopes of a target antigen. In some embodiments, one of the binding specificities is for one target antigen, which is not an antibody or antibody mimetic, and the other is for a second target antigen, which is an antibody or antibody mimetic.
  • the target antibody or antibody mimetic recognized by an anti-idiotype polypeptide includes an Fc domain from IgM, IgD, IgG, IgA, or IgE.
  • the antibody or antibody mimetic recognized by an anti-idiotype polypeptide includes an Fc domain from IgM, IgD, IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, or IgE.
  • a target antibody or antibody mimetic with an IgM Fc domain can be used to drive higher order multimerization.
  • the target antibody or antibody mimetic includes glycosylated residues. In some embodiments, the target antibody or antibody mimetic includes an a-Gal epitope. In some embodiments, the target antibody or antibody mimetic includes one or more glycoforms. In some embodiments, the glycoforms or antibody mimetic includes the target antibody with an a-l,3-Gal residue. In some embodiments, the glycoforms comprise the target antibody with an N-glycolylneuraminic acid residue. In some embodiments, the glycoforms comprise the target antibody with an oligomannose.
  • the cell line an antibody or antibody mimetic is produced in can affect glycosylation of the residues in the polypeptide.
  • the target antibody or antibody mimetic can be produced in a CHO cell line, a BHK cell line, an NS0 cell line, an SP2/0 cell line, a YB2/0 cell line, or an HEK293 cell line or its derivatives (e.g., a HT-1080 cell line, a Huh-7 cell line, or a PER.C6 cell line).
  • the target antibody or antibody mimetic can be produced in a CHO cell line.
  • the target antibody or antibody mimetic can be produced in an NSO cell line.
  • the target antibody or antibody mimetic can be produced in an SP2/0 cell line.
  • the anti-idiotype polypeptide recognizes a target antibody mimetic.
  • the target antibody mimetic can be an afflilin, an affimer, an affitin, an alphabody, an alphamab, an anticalin, a peptide aptamer, an armadillo repeat protein, an atrimer, an avimer (also known as avidity multimer), a C-type lectin domain, a cysteine-knot miniprotein, a cyclic peptide, a cytotoxic T- lymphocyte associated protein-4, a DARPin (Designed Ankyrin Repeat Protein), a fibrinogen domain, a fibronectin binding domain (FN3 domain) (e.g., adnectin or monobody), a fynomer, a knottin, a Kunitz domain peptide, a nanofitin, a leucine-rich repeat domain, a lipocalin domain, a
  • the amino acids in the polypeptide sequences of the extracellular recognition domain of an anti-idiotype polypeptide or of the antibodies and antibody mimetics to which the anti-idiotype polypeptide binds can contain substitutions or variations.
  • the antiidiotype polypeptides, antibodies and antibody mimetics obtained by performing substitutions or variations in the amino acid sequences can be referred to as anti-idiotype polypeptide variants, antibody variants and antibody mimetic variants, respectively.
  • the antibodies and antibody mimetics to which the anti-idiotype polypeptide binds can be antibody variants and antibody mimetic variants, respectively.
  • Anti-idiotype polypeptide variants, antibody variants, and antibody mimetic variants can be prepared by introducing appropriate changes into the nucleotide sequence of the nucleic acids encoding the antibody or antibody mimetic.
  • antibody variants and antibody mimetic variants can be prepared by peptide synthesis. Such modifications include, for example, deletions, insertions, and/or substitutions of residues within the amino acid sequences of the anti-idiotype polypeptide, antibody, or antibody mimetic.
  • any combination of deletion, insertion, and substitution can be made to generate the anti-idiotype polypeptide variant, antibody variant, or antibody mimetic variant, provided that the anti-idiotype polypeptide variant, antibody variant, or antibody mimetic variant possesses the desired characteristics, for example, idiotype or antigen binding.
  • the variations can be introduced into the antibody in order to improve the binding affinity, and/or other biological properties of the antibody.
  • the variants include one or more amino acid substitutions.
  • an alanine (Ala) residue can be substituted with valine (Vai), leucine (Leu), or isoleucine (He).
  • an arginine (Arg) residue can be substituted with lysine (Lys), glutamine (Gin), or asparagine (Asn).
  • an asparagine (Asn) residue can be substituted with glutamine (Gin), histidine (His), aspartic acid (Asp), lysine (Lys), or arginine (Arg).
  • an aspartic acid (Asp) residue can be substituted with glutamic acid (Glu) or asparagine (Asn).
  • a cysteine (Cys) residue can be substituted with serine (Ser) or alanine (Ala).
  • a glutamine (Gin) residue can be substituted with asparagine (Asn) or glutamic acid (Glu).
  • a glutamic acid (Glu) residue can be substituted with aspartic acid (Asp) or glutamine (Gin).
  • a glycine (Gly) residue can be substituted with alanine (Ala).
  • a histidine (His) residue can be substituted with asparagine (Asn), glutamine (Gin), lysine (Lys), or arginine (Arg).
  • an isoleucine (He) residue can be substituted with leucine (Leu), valine (Vai), methionine (Met), alanine (Ala), phenylalanine (Phe), or norleucine.
  • a leucine (Leu) residue can be substituted with norleucine, isoleucine (He), valine (Vai), methionine (Met), alanine (Ala), or phenylalanine (Phe).
  • a lysine (Lys) residue can be substituted with arginine (Arg), glutamine (Gin), or asparagine (Asn).
  • a methionine (Met) residue can be substituted with leucine (Leu), phenylalanine (Phe), or isoleucine (He).
  • a phenylalanine (Phe) residue can be substituted with tryptophan (Trp), leucine (Leu), valine (Vai), isoleucine (He), alanine (Ala), or tyrosine (Tyr).
  • a proline residue can be substituted with alanine (Ala).
  • a serine (Ser) residue can be substituted with threonine (Thr).
  • a threonine (Thr) residue can be substituted with valine (Vai) or serine (Ser).
  • a tryptophan (Trp) can be substituted with tyrosine (Tyr) or phenylalanine (Phe).
  • a tyrosine (Tyr) residue can be substituted with tryptophan (Trp), phenylalanine (Phe), threonine (Thr), or serine (Ser).
  • a valine (Vai) residue can be substituted with isoleucine (He), leucine (Leu), methionine (Met), phenylalanine (Phe), alanine (Ala), or norleucine.
  • the target antibody or antibody mimetic can include an Fc region.
  • glycosylation of the Fc region of the target antibody or antibody mimetic can affect the antibody effector function.
  • the antibody or antibody mimetic including an Fc region can be altered by altering the carbohydrate attached to the Fc region.
  • Native antibodies produced by mammalian cells typically include a branched, biantennary oligosaccharide that is typically attached by an N-linkage to Asn297 of the CH2 domain of the Fc region.
  • the oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure.
  • modifications of the oligosaccharide in an antibody or antibody mimetic of the invention can be made to generate antibody or antibody mimetic variants with certain improved properties.
  • antibody or antibody mimetic variants include a carbohydrate structure that lacks fucose directly or indirectly attached to an Fc region.
  • the amount of fucose in the antibody or antibody mimetic variants may be from 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%.
  • the amount of fucose can be determined by calculating the average amount of fucose within the sugar chain at Asn 297, relative to the sum of all glycostructures attached to Asn 297 (e.g., complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in W02008/077546, for example.
  • Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 can also be located within about 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, for example, US Patent Publication Nos. US2003/0157108; US2004/0093621.
  • the antibody or antibody mimetic variants can include bisected oligosaccharides in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by N-acetylglucosamine (GlcNAc).
  • the bisecting GlcNAc structure is a [31,4-linked GlcNAc attached to the core [3-mannose residue, representing a special type of N-glycosylated modification.
  • GlcNAc transferred to the 4-positioii of fee p-linked core mannose (Man) residue in complex or hybrid N- glycans by the pi.4-mannosyl-glycoprotein 4-p-N-acetylglucosaniinyltransferase (GlcNAc -T III) is considered as a bisecting structure that is usually not considered as an antenna because it cannot be further extended by the proper enzymes.
  • Such antibody and antibody mimetic variants can have reduced fucosylation and/or improved ADCC antibody effector function.
  • antibody or antibody mimetic variants can include at least one galactose residue in the oligosaccharide attached to the Fc region.
  • the Fc region of an antibody or antibody mimetic can include modifications, e.g., substitutions, in one or more amino acids, thereby generating an Fc region variant.
  • the Fc region variant can include a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region).
  • an antibody or antibody mimetic variant includes an Fc region with one or more amino acid substitutions can have improved ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region of SEQ ID NO:698 (Heavy chain of E27 Anti-IgE antibody).
  • an antibody or antibody mimetic variant includes a modification in the Fc region that results in altered (i.e., either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC). ).
  • CDC Complement Dependent Cytotoxicity
  • an antibody or antibody mimetic disclosed herein can be derivatized with non-proteinaceous moieties that are known in the art.
  • the non-proteinaceous moieties suitable for derivatization of the antibody or antibody mimetic include but are not limited to water-soluble polymers.
  • water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly- 1,3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, poly oxyethylated polyols (e.g., glycerol), and/or polyvinyl alcohol, and combinations thereof.
  • PEG polyethylene glycol
  • the polymer can be of any molecular weight, and can be branched or unbranched.
  • the number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody or antibody mimetic to be improved, and whether the antibody or antibody mimetic derivative will be used in a therapy under defined conditions.
  • the antibody- or antibody mimetic-polymer conjugates can be made using any suitable technique for derivatizing antibodies with polymers.
  • the antibody- and antibody mimetic-polymer conjugates as disclosed herein include species wherein a polymer is covalently attached to a specific site or specific sites on the parental antibody, i.e., polymer attachment is targeted to a particular region or a particular amino acid residue or residues in the parental antibody or antibody mimetic.
  • Site specific conjugation of polymers can be performed by attachment to cysteine residues in the parental antibody or antibody mimetic.
  • the coupling chemistry can, for example, utilize the free sulfhydryl group of a cysteine residue not in a disulfide bridge in the parental antibody.
  • the polymer can be activated with any functional group capable of reacting specifically with the free sulfhydryl or thiol group(s) on the parental antibody, such as maleimide, sulfhydryl, thiol, triflate, tesylate, aziridine, exirane, and 5-pyridyl functional groups.
  • the polymer can be coupled to the parental antibody using any protocol suitable for the chemistry of the coupling system selected..
  • one or more cysteine residues naturally present in the parental antibody or antibody mimetic are used as attachment sites for polymer conjugation.
  • one or more cysteine residues are engineered into a selected site or sites in the parental antibody or antibody mimetic for the purpose of providing a specific attachment site or sites for polymer.
  • antibody fragments such as Fab
  • Fab can be derivatized to form antibody fragment-polymer conjugates, and the polymer is attached to one or more cysteine residue in the light or heavy chain of the fragment that would ordinarily form the inter-chain disulfide bond linking the light and heavy chains.
  • the domains of the antibodies and/or antibody mimetics bound by the anti-idiotype polypeptides can control or modulate the downstream effects the anti-idiotype polypeptides have upon binding to their target antibodies or target antibody mimetics.
  • Fc domains of an antibody can change the downstream effects of the antibody binding to an antigen.
  • the Fc domain of the antibody can also affect the function of the antibody binding to the anti-idiotype polypeptide, also referred to herein as the antibody effector function.
  • the target antibody or antibody mimetic includes an Fc domain that is capable of cross-linking with the Fc receptors on an effector cell to initiate antibody-dependent cellular cytotoxicity (ADCC) leading to death of the cells that are bind the target antibody or antibody mimetic.
  • the effector cell may be one or more of NK cells, monocytes, macrophages, and granulocytes.
  • the Fc domain variants include five isotypes, IgM, IgD, IgG, IgA, and IgE, each with unique structural features that impact the antibody function.
  • the IgG isotype is further divided into four subclasses, i.e., IgGl, IgG2, IgG3, and IgG4, and the IgA isotype is further divided into two subclasses, i.e., IgAl and IgA2.
  • the antibody recognized by an anti-idiotype polypeptide includes an Fc domain from IgM, IgD, IgG, IgA, or IgE.
  • the antibody recognized by an anti-idiotype polypeptide includes an Fc domain from IgM, IgD, IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, or IgE. In some embodiments, the antibody recognized by an antiidiotype polypeptide includes an Fc domain from IgGl, IgG2, IgG3, or IgG4. In some embodiments, the antibody recognized by an anti-idiotype polypeptide includes an Fc domain from IgAl or IgA2. In some embodiments, the Fc domain of the antibody recognized by an anti-idiotype polypeptide is or is derived from the Fc domain of an antibody from any animal.
  • the Fc domain of the antibody recognized by an anti-idiotype polypeptide is or is derived from a rat, mouse, or in illustrative embodiments a human Fc domain and retains at least 85%, 90%, 95%, or 99% sequence identity.
  • the Fc domain of the antibody recognized by an anti-idiotype polypeptide can be chimeric IgGl, human IgGl, human IgG2, human IgG4, human IgM, humanized IgGl, humanized IgG2, humanized IgG2/4, humanized IgG4, mouse IgGl, or mouse IgG2a.
  • the anti-idiotype polypeptide induces, promotes, or activates antibody effector functions upon antibody or antibody mimetic binding to the anti-idiotype extracellular recognition domain of the anti-idiotype polypeptide expressed on the surface of the cell.
  • binding of the antibody or antibody mimetic to the anti-idiotype polypeptide induces, promotes, or activates one or more of antibody-mediated complement activation, antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis, antibody-dependent enhancement of diseases, and opsonization.
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • binding of the antibody or antibody mimetic to the anti-idiotype polypeptide does not induce, promote, or activate ADCC.
  • the antibody includes an Fc domain derived from IgM, IgG, or IgA and binding of the anti-idiotype polypeptide to the antibody induces, promotes, or activates one or more of macrophage opsonophagocytosis, oxidative burst or release of cytokines, and antimicrobial peptides.
  • the antibody includes an Fc domain derived from IgM, IgG, or IgA and binding of the anti-idiotype polypeptide to the antibody induces, promotes, or activates one or more of antigen uptake, DC maturation, and antigen presentation.
  • the antibody includes an Fc domain derived from IgM, IgG, or IgA and binding of the anti-idiotype polypeptide to the antibody induces, promotes, or activates antigen capture on follicular dendritic cells for presentation to B cells.
  • the antibody includes an Fc domain derived from IgD, IgG, or IgE and binding of the anti-idiotype polypeptide to the antibody induces, promotes, or activates granulocyte degranulation and release of one or more of vasoactive mediators, chemoattractants and Tn2-type cytokines.
  • the antibody includes an Fc domain derived from IgM or IgG and binding of the antiidiotype polypeptide to the antibody induces, promotes, or activates complement-mediated production of chemoattractants, cytotoxicity, and opsonophagocytosis.
  • the antibody includes an Fc domain derived from IgM or IgG and binding of the anti-idiotype polypeptide to the antibody induces, promotes, or activates one or more of neutrophil activation, opsonophagocytosis, oxidative burst, and induction of neutrophil extracellular baps.
  • the antibody includes an Fc domain derived from IgG and binding of the anti-idiotype polypeptide to the antibody induces, promotes, or activates one or more of NK cell degranulation and cytotoxicity.
  • an anti-idiotype polypeptide that includes an extracellular recognition domain and a membrane association domain, wherein the extracellular recognition domain comprises a domain that recognizes an idiotype of a target antibody or a target antibody mimetic.
  • a polynucleotide encoding the anti-idiotype polypeptide is a polynucleotide that includes one or more transcriptional units, wherein each of the one or more transcriptional units is operatively linked to a promoter wherein the one or more transcriptional units comprise: a) nucleic acids a polynucleotide sequence encoding one or more inhibitory RNA molecules and/or a first engineered signaling polypeptide, and b) nucleic acids a polynucleotide sequence encoding an anti-idiotype polypeptide comprising an extracellular recognition domain and a membrane association domain, wherein the extracellular recognition domain comprises a domain that recognizes an idiotype of a target antibody or antibody mimetic.
  • the anti-idiotype polypeptide further includes one or more of a stalk domain, an intracellular domain, and/or a linker.
  • the anti-idiotype polypeptide can also be part a fusion polypeptide, as disclosed in more detail below.
  • the membrane association domain can be part of the polypeptide to which the anti-idiotype polypeptide is fused.
  • the anti-idiotype polypeptide can be at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 amino acids in length.
  • the anti-idiotype polypeptide can be between 10 and 1000, 10 and 900, 10 and 800, 10 and 700, 10 and 600, 10 and 500, 10 and 400, 10 and 300, 10 and 200, 10 and 100, 10 and 50, 25 and 1000, 25 and 900, 25 and 800, 25 and 700, 25 and 600, 25 and 500, 25 and 400, 25 and 300, 25 and 200, 25 and 100, 25 and 50, 50 and 1000, 50 and 900, 50 and 800, 50 and 700, 50 and 600, 50 and 500, 50 and 400, 50 and 300, 50 and 200, 50 and 100, 100 and 1000, 100 and 900, 100 and 800, 100 and 700, 100 and 600, 100 and 500, 100 and 400, 100 and 300, or 100 and 200 amino acids in length.
  • the anti-idiotype extracellular recognition domain of an anti-idiotype polypeptide is typically all or more typically part of an anti-idiotype antibody or anti-idiotype antibody mimetic.
  • the extracellular recognition domain of an anti-idiotype polypeptide is capable of, has a structure for, is designed to, is selected for, is effective for, and/or is adapted for, recognizing, binding to, or otherwise interacting with the idiotype of a target antibody or the idiotype of a target antibody mimetic.
  • a recognition domain of an anti-idiotype antibody or antibody mimetic, and the target antibody or antibody mimetic that has the idiotype to which it binds are specific binding pair members.
  • an extracellular recognition domain of an anti-idiotype polypeptide recognizes the idiotype of any antibody or antibody mimetic known in the art, which can be, for nonlimiting example, a clinical antibody or clinical antibody mimetic, as discussed in further detail herein.
  • the extracellular recognition domain of an anti-idiotype polypeptide herein includes an idiotype-binding variable region of an anti-idiotype antibody or an idiotype-binding region of an antiidiotype antibody mimetic.
  • Such variable region can include a framework that is derived from a human framework region.
  • Such anti-idiotype antibody or antibody mimetic can be any of antibody fragment or any type of antibody mimetic provided herein.
  • the extracellular recognition domain can be an antibody such as a full-length antibody, a single-chain antibody, a Fab fragment, a Fab' fragment, a (Fab')2 fragment, a Fv fragment, an scFv, a divalent single-chain antibody, a diabody, a chimeric antibody, or a disbud.
  • the extracellular recognition domain is a single chain Fv (scFv).
  • the heavy chain is positioned N-terminal of the light chain in the engineered signaling polypeptide. In other embodiments, the light chain is positioned N-terminal of the heavy chain in the engineered signaling polypeptide.
  • the heavy and light chains can be separated by a linker as discussed in more detail herein.
  • the heavy or light chain can be at the N-terminus of the engineered signaling polypeptide and is typically C-terminal of another domain, such as a signal sequence or peptide.
  • the extracellular recognition domain can include one or more domains from or derived from a mouse antibody, a rat antibody, a rabbit antibody, a goat antibody, a chicken antibody, a sheep antibody, a cow antibody, or a llama antibody, or in illustrative embodiments, a human antibody.
  • Other antibody-based recognition domains cAb VHH (camelid antibody variable domains) and humanized versions, IgNAR VH (shark antibody variable domains) and humanized versions, sdAb VH (single domain antibody variable domains) and "camelized" antibody variable domains are suitable for use as extracellular recognition domains of anti-idiotype polypeptides provided herein, and methods using the same.
  • T cell receptor (TCR) based recognition domains can be the extracellular recognition domains.
  • the extracellular recognition domain can be bispecific or include domains from a bispecific antibody.
  • the extracellular recognition domain can be multispecific or include domains from a multispecific antibody.
  • Multispecific antibodies have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for one target antibody or antibody mimetic and the other is for another target antibody or antibody mimetic.
  • bispecific antibodies may bind to two different epitopes of a target antibody or antibody mimetic.
  • the extracellular recognition domain can include a recognition domain of an anti-idiotype antibody or antibody mimetic, and also include an ASTR of any of the CARs disclosed herein.
  • the extracellular recognition domain can be at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300 amino acids in length. In some embodiments, the extracellular recognition domain can be between 10 and 500, 10 and 400, 10 and 300, 10 and 200, 10 and 100, 10 and 50, 10 and 30, 20 and 500, 20 and 400, 20 and 300, 20 and 200, 20 and 100, 20 and 50, 20 and 30, 50 and 500, 50 and 400, 50 and 300, 50 and 200, 50 and 100, 100 and 500, 100 and 400, 100 and 300, 100 and 200, 125 and 275, 150 and 275, or 150 and 250 amino acids in length.
  • Methods for making anti-idiotype extracellular recognition domains can utilize known methods for making anti-idiotype antibodies and antibody mimetics, applying a strategy, for example that is provided in the Examples herein for an exemplary phage display strategy.
  • Such methods can include makine a library of antibodies or antibody mimetics using virtually any method known in the art for creating such library, and screening the library for binding to the idiotype of a target antibody.
  • the library can be constructed by varying residues of an existing anti-idiotype antibody, or can involve a general antibody or antibody mimetic library that does not start with a selected prior anti-idiotype antibodies. Once the library is designed and constructed, the translated antibody or antibody mimetic protein scaffolds are screened for isolation of mutants with the desired properties.
  • the most commonly employed display systems include phage display, ribosome display, mRNA display, yeast display, and bacterial cell-surface display (Lipovsek and Pliickthun, J Immunol Methods. 2004 Jul;290(l-2):51-67).
  • phage display a display of phage
  • ribosome display a display of ribosomes
  • mRNA display a display of ribosomes
  • yeast display bacterial cell-surface display
  • a skilled artisan will understand how to use this and similar systems to identify appropriate anti-idiotype antbodies and antibody mimetics, typically using all or a portion of the target antibody as bate, which can be present on a solid support such as a bead during the screening. Screening can occur over several rounds, where conditions can be varied to improve isolated library members for one or more characteristics, including affinity for the idiotype.
  • binding of the anti-id ERD of the anti-idiotype polypeptide to its target antibody or antibody mimetic prevents and/or blocks binding of the target antibody or antibody mimetic to the cognate antigen of the target antibody or antibody mimetic.
  • the extracellular recognition domain of the anti-idiotype polypeptide is capable of, adapted for, and/or configured to prevent and/or block binding of the target antibody or antibody mimetic to the cognate antigen of the target antibody or antibody mimetic when the anti-idiotype polypeptide is bound to the target antibody or antibody mimetic.
  • the target antibody is cetuximab and binding of the extracellular recognition domain of the anti-idiotype polypeptide to cetuximab prevents and/or blocks binding of cetuximab to Epidermal Growth Factor Receptor (EGFR).
  • the extracellular recognition domain of the anti-idiotype polypeptide is capable of, adapted for, and/or configured to prevent and/or block binding of cetuximab to EGFR when the anti-idiotype polypeptide is bound to cetuximab.
  • binding of the anti-idiotype polypeptide to the target antibody does not block or prevent binding between the target antibody and its cognate antigen.
  • binding of the anti-idiotype polypeptide to the target antibody blocks or prevents binding between the target antibody and its cognate antigen.
  • the extracellular recognition domain recognizes the antigen-binding site of the target antibody or the antibody mimetic. In some embodiments, the extracellular recognition domain recognizes the variable region but does not recognize the antigen-binding site of the target antibody or the antibody mimetic. In some embodiments, the extracellular recognition domain recognizes a portion of the variable region that is not a part of the antigen-binding site of the target antibody or the antibody mimetic.
  • binding of the extracellular recognition domain to the target antibody or antibody mimetic prevents and/or blocks binding of the target antibody or antibody mimetic to the cognate antigen of the target antibody or antibody mimetic.
  • the antigen-binding site can include the residues important for the target antibody or antibody mimetic to recognize its cognate antigen.
  • the anti-id ERD recognizes (e.g., is capable of binding to) cetuximab and comprises any of the sequences provided in this paragraph.
  • the anti-id ERD can be encoded by a polynucleotide with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all the polynucleotides of one or more of SEQ ID NOs:376-436.
  • the anti-id ERD can include a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all the amino acids of one or more of SEQ ID NOs:437-497.
  • the extracellular recognition domain can include a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids of one or more of SEQ ID NOs:498-599.
  • the extracellular recognition domain can include a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids of one or more of SEQ ID NOs:600-672.
  • the anti-id ERD recognizes (e.g., is capable of binding to) cetuximab and comprises any of the sequences provided in this paragraph.
  • the HCDR1, HCDR2, and HCDR3 of an extracellular recognition domain can include any of the following combinations of SEQ ID NOs:498, 528, and 560; 499, 529, and 561; 500, 530, and 562; 501, 531, and 563; 502, 532, and
  • the anti-id ERD recognizes (e.g., is capable of binding to) cetuximab and comprises any of the sequences provided in this paragraph.
  • the LCDR1, LCDR2, and LCDR3 of an extracellular recognition domain can include any of the following combinations of SEQ ID NOs:600, 625, and 634; 601, 625, and 635; 600, 625, and 636; 602, 625, and 637; 603, 626, and 638; 603, 626, and 639; 604, 630, and 640; 605, 628, and 641; 606, 625, and 642; 603, 626, and 643; 607, 632, and 644; 608, 625, and 645; 609, 625, and 646; 610, 630, and 647; 604, 630, and 648; 611, 625, and 649; 606, 625, and 650; 612, 625, and 649; 607, 632
  • the anti-id ERD recognizes (e.g., is capable of binding to) cetuximab and comprises any of the sequences provided in this paragraph.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of an extracellular recognition domain can include any of the following combinations of SEQ ID NOs: 498, 528, 560, 600, 625, and 634; 499, 529, 561, 601, 625, and 635; 500, 530, 562, 600, 625, and 636; 501, 531, 563, 602, 625, and 637; 502, 532, 564, 603, 626, and
  • polypeptide sequences at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids of any of the preceding SEQ ID NOs.
  • the extracellular recognition domain can be from or derived from an antiidiotype antibody mimetic.
  • the anti-idiotype antibody mimetic can be an afflilin, an affimer, an affitin, an alphabody, an alphamab, an anticalin, a peptide aptamer, an armadillo repeat protein, an atrimer, an avimer (also known as avidity multimer), a C-type lectin domain, a cysteine-knot miniprotein, a cyclic peptide, a cytotoxic T-lymphocyte associated protein-4, a DARPin (Designed Ankyrin Repeat Protein), a fibrinogen domain, a fibronectin binding domain (FN3 domain) (e.g., adnectin or monobody), a fynomer, a knottin, a Kunitz domain peptide, a nanofitin, a leucine -rich repeat domain, a lip
  • the extracellular recognition domain of the anti-idiotype polypeptide includes an affimer that is capable of binding to a target antibody.
  • the average size of the affimer can be in the range of 12-14 kDa.
  • the affimers capable of binding to a specific target is designed by selecting an appropriate scaffold that is suitable for incorporation of recognition domains.
  • the scaffold is a three-dimensional protein structure that is suitable for mutations and insertions with enough flexibility in its primary structure that the introduced modifications do not compromise its secondary structure and overall stability.
  • the scaffolds are typically small, thermostable, single-domain proteins without any disulfide bonds or glycosylation.
  • Affimers can be made from two scaffolds, Adhiron scaffolds and human stefin A scaffolds.
  • the stefin A scaffold is engineered from human stefin A protein, whereas the Adhiron scaffold is synthetic, originally based on the sequence of cystatin.
  • the library is designed by employing in silico methods and is constructed via molecular biology protocols. Once the possible candidates for mutagenesis are identified, mutant constructs and libraries of the antibody mimetics can be generated at the DNA level by employing either site directed or random mutagenesis strategies.
  • the translated protein scaffolds are screened for isolation of mutants with the desired properties.
  • the most commonly employed display systems include phage display, ribosome display, mRNA display, yeast display, and bacterial cellsurface display. A skilled artisan will understand how to use this and similar systems to identify appropriate anti-idiotype affimers.
  • the extracellular recognition domain of the anti-idiotype polypeptide includes a DARPin that is capable of binding to a target antibody.
  • DARPins are made from tightly packed repeats of 33 amino acid residues. Each repeat forms a structural unit consisting of a [3-turn followed by two antiparallel a-helices.
  • DARPins are small proteins having a molecular weight in the range of 14-18 kDa that are extremely thermostable and resistant to proteases and denaturing agents.
  • DARPins usually have a scaffold that is a constant region, and have variable sites in which amino acid substitutions do not alter the protein conformation.
  • the process of designing scaffolds involves the design of a library of protein variants by random site-specific mutagenesis; and selection of molecules using techniques like yeast display, phage display, and ribosome display.
  • DARPins can recognize targets with high specificities and affinities, surpassing that of the antibodies. A skilled artisan will understand how to use this and similar systems to identify appropriate anti-idiotype DARPins.
  • the extracellular recognition domain of the anti-idiotype polypeptide includes a nanobody that is capable of binding to a target antibody.
  • a nanobody is an antigen-binding fragment, with a size of approximately 12-15 kDa.
  • the nanobody includes the antigen-binding capacity of the original heavy chain antibodies, evolved to be fully functional in the absence of a light chain.
  • Nanobodies consist of three antigenic complementary determining regions (CDRs) and four frame regions (FRs). The CDRs are the binding regions of nanobody to the antigen or a target antibody.
  • CDRs complementary determining regions
  • FRs frame regions
  • Phage display can be used to screen and enrich nanobody-phage with specific binding ability from the nanobody library. Functional verification and confirmation of the nanobody expression can also be performed to select the optimal candidate. A skilled artisan will understand how to use this and similar systems to identify appropriate anti-idiotype nanobodies.
  • the anti-idiotype polypeptide includes a stalk which is located in the portion of the anti-idiotype polypeptide lying outside the cell and interposed between the extracellular recognition domain and the membrane association domain.
  • the stalk has at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to a wild-type CD8 stalk region (TTTPAPRPPTPAPTIASQPESERPEACRPAAGGAVHTRGEDFA (SEQ ID NO:2), has at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to a wild-type CD28 stalk region (FCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO:3)), or has at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to a wild-type immunoglobulin heavy chain stalk region.
  • the stalk employed allows the extracellular recognition domain, and typically
  • the stalk region can have a length of from about 4 amino acids to about 50 amino acids, e.g., from about 4 aa to about 10 aa, from about 10 aa to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 40 aa, or from about 40 aa to about 50 aa.
  • the stalk of an anti-idiotype polypeptide includes at least one cysteine.
  • the stalk can include the sequence Cys-Pro-Pro-Cys (SEQ ID NO:4). If present, a cysteine in the stalk of a first anti-idiotype polypeptide can be available to form a disulfide bond with a stalk in a second anti-idiotype polypeptide.
  • Stalks can include immunoglobulin hinge region amino acid sequences that are known in the art; see, e.g., Tan et al. (1990) Proc. Natl. Acad. Sci. USA 87:162; and Huck et al. (1986) Nucl. Acids Res. 14:1779.
  • an immunoglobulin hinge region can include a domain with at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids of any of the following amino acid sequences: DKTHT (SEQ ID NO:5); CPPC (SEQ ID NO:4); CPEPKSCDTPPPCPR (SEQ ID NO:6) (see, e.g., Glaser et al. (2005) J. Biol. Chem.
  • ELKTPLGDTTHT SEQ ID NO:7
  • KSCDKTHTCP SEQ ID NO:8
  • KCCVDCP SEQ ID NO:9
  • KYGPPCP SEQ ID NO: 10
  • EPKSCDKTHTCPPCP SEQ ID NO: 11
  • ERKCCVECPPCP SEQ ID NO: 12
  • ELKTPLGDTTHTCPRCP SEQ ID NO: 13
  • SPNMVPHAHHAQ SEQ ID NO: 14
  • the stalk can include a hinge region with an amino acid sequence of a human IgGl, IgG2, IgG3, or IgG4, hinge region.
  • the stalk can include one or more amino acid substitutions and/or insertions and/or deletions compared to a wild-type (naturally-occurring) hinge region.
  • His229 of human IgG 1 hinge can be substituted with Tyr, so that the stalk includes the sequence EPKSCDKTYTCPPCP (SEQ ID NO:15), (see, e.g., Yan et al. (2012) J. Biol. Chem. 287:5891).
  • the stalk can include an amino acid sequence derived from human CD8; e.g., the stalk can include the amino acid sequence:
  • TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD SEQ ID NO: 16
  • the stalk domain of an anti-idiotype polypeptide can include any of the dimerizing moieties disclosed herein.
  • the dimerizing motif can be selected from the group consisting of: a leucine zipper motif-containing polypeptide, CD69, CD71, CD72, CD96, Cdl05, Cdl61, Cdl62, Cd249, CD271, and Cd324, as well as mutants and/or active fragments thereof that retain the ability to dimerize.
  • the membrane association domain of an anti-idiotype polypeptide attaches the extracellular recognition domain of the anti-idiotype polypeptide to a cell membrane.
  • the membrane association is a transmembrane domain.
  • the transmembrane domain is a heterologous transmembrane domain.
  • the transmembrane domain is an endogenous transmembrane domain.
  • the transmembrane domain is from or derived from an antibody.
  • the transmembrane domain is from or derived from IgD.
  • the polynucleotide, vector, or cell can further include nucleic acids encoding IgA and IgB.
  • the transmembrane domain is from or derived from BAFFR, C3Z, CEACAM1, CD2, CD3A, CD3B, CD3D, CD3E, CD3G, CD3Z, CD4, CD5, CD7, CD8A, CD8B, CD9, CD11A, CD11B, CD11C, CD11D, CD27, CD16, CD18, CD19, CD22, CD28, CD29, CD33, CD37, CD40, CD45, CD49A, CD49D, CD49F, CD64, CD79A, CD79B, CD80, CD84, CD86, CD96 (Tactile), CD100 (SEMA4D), CD103, C134, CD137, CD154, CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (Ly9), CD247, CRLF2, CRTAM, CSF2RA, CSF2RB, CSF3R, EPOR, FCER1G, FCGR2
  • the transmembrane domain includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids of one or more of the following: CD8 alpha TM (SEQ ID NO:17); CD8 beta TM (SEQ ID NO:18); CD4 stalk (SEQ ID NO:19); CD3Z TM (SEQ ID NO:20); CD28 TM (SEQ ID NO:21); CD134 (0X40) TM: (SEQ ID NO:22); CD7 TM (SEQ ID NO:23); CD8 stalk and TM (SEQ ID NO:24); CD28 stalk and TM (SEQ ID NO:25); the hPDGFRb hinge and TM of SEQ ID NO:676 (this sequence also includes an 8 aa intracellular domain); the CD28 hinge and TM of SEQ ID NO:677 (this sequence also includes 99% or 100% sequence
  • the transmembrane domain of an anti-idiotype polypeptide can include any of the dimerizing moieties disclosed herein.
  • the dimerizing motif can be selected from the group consisting of: a leucine zipper motif-containing polypeptide, CD69, CD71, CD72, CD96, Cdl05, Cdl61, Cdl62, Cd249, CD271, and Cd324, as well as mutants and/or active fragments thereof that retain the ability to dimerize.
  • the membrane association domain is glycosylphosphatidylinositol (GPI).
  • the anti-idiotype polypeptide is fused to a lymphoproliferative element, CAR, and/or recombinant TCR, and the membrane association domain can be the transmembrane domain of the lymphoproliferative element, the CAR, and/or the recombinant TCR.
  • the intracellular domain of an anti-idiotype polypeptide is typically all or, more typically, part of one or more intracellular domains known in the art or disclosed herein.
  • the intracellular domain of an anti-idiotype polypeptide can perform various functions, including both non-signaling functions, for example, to anchor the anti-idiotype polypeptide, and signaling functions, for example, to activate proliferative, survival, and/or cell death signaling or modulate transcriptional activity.
  • an anti-idiotype polypeptide includes an intracellular domain.
  • the intracellular domain of the fusion polypeptide can be the anti-idiotype polypeptide intracellular domain.
  • the intracellular domain of the fusion polypeptide can be the intracellular domain of the polypeptide to which the anti-idiotype polypeptide is fused, for example, the intracellular domain of a lymphoproliferative element, a CAR, and/or a recombinant TCR.
  • the intracellular domains that can be used include any of the intracellular domains of lymphoproliferative elements, CARs, and recombinant TCRs are disclosed elsewhere herein.
  • the intracellular domain of an anti-idiotype polypeptide can include one or more, two or more, or three or more intracellular domains from other polypeptides, for example any of the intracellular domains of a lymphoproliferative element, a CAR, and/or a recombinant TCR disclosed herein.
  • the intracellular domain can include one or more of any of the intracellular activating domains, modulatory domains, or intracellular signaling domains disclosed elsewhere herein.
  • the intracellular domain of an anti-idiotype polypeptide can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 35, 40 45, 50, 75, 100, 125, 150, 175, or 200 amino acids in length.
  • the intracellular domain of an anti-idiotype polypeptide can be between 1 and 1000, 1 and 900, 1 and 800, 1 and 700, 1 and 600, 1 and 500, 1 and 400, 1 and 300, 1 and 200, 1 and 100, 1 and 50, 1 and 25, 5 and 1000, 5 and 900, 5 and 800, 5 and 700, 5 and 600, 5 and 500, 5 and 400, 5 and 300, 5 and 200, 5 and 100, 5 and 50, 5 and 25, 10 and 1000, 10 and 900, 10 and 800, 10 and 700, 10 and 600, 10 and 500, 10 and 400, 10 and 300, 10 and 200, 10 and 100, 10 and 50, 10 and 25, 25 and 1000, 25 and 900, 25 and 800, 25 and 700, 25 and 600, 25 and 500, 25 and 400, 25 and 300, 25 and 200, 25 and 100, 25 and 50, 50 and 1000, 50 and 900, 50 and 800, 50 and 700, 50 and 600, 50 and 500, 50 and 400, 50 and 300, 50 and 200, 50 and 100, 100, 25 and 50, 50 and
  • the intracellular domain is an anchor for the anti-idiotype polypeptide. In some embodiments, the intracellular domain has no signaling activity. [0182] In some embodiments, signaling through the intracellular domains of the anti-idiotype polypeptide is inducible, and signaling can be activated by addition of the target antibody or antibody mimetic. Such anti-idiotype polypeptides can be referred to herein as activity modulators, where upon addition of the target antibody or antibody mimetic, two or more anti-idiotype polypeptides can dimerize through binding the same molecule of target antibody or antibody mimetic.
  • This dimerization of the antiidiotype polypeptides dimerizes the intracellular domains of the two anti-idiotype polypeptides, resulting in activation based if the appropriate intracellular domains are used.
  • the intracellular domains of an activity modulating anti-idiotype polypeptide can be activated by dimerization and the only dimerizing moiety on the anti-idiotype polypeptide is the extracellular recognition domain.
  • the intracellular domains of an activity modulating anti-idiotype polypeptide can be activated by dimerization and the anti-idiotype polypeptide includes a separate inducible or constitutive dimerizing moiety on the anti-idiotype polypeptides besides the extracellular recognition domain.
  • the intracellular domains of an activity modulating anti-idiotype polypeptide can be activated by multimerization of at least three domains (e.g., trimerization) and the only dimerizing moiety on the anti-idiotype polypeptide is the extracellular recognition domain.
  • the intracellular domains of an activity modulating anti-idiotype polypeptide can be activated by multimerization of at least three domains (e.g., trimerization) and the anti-idiotype polypeptide includes a separate inducible or constitutive dimerizing moiety on the anti-idiotype polypeptides besides the extracellular recognition domain.
  • the intracellular domains of an activity modulating anti-idiotype polypeptide that are activated by multimerization of at least three domains include a separate constitutive dimerizing moiety on the antiidiotype polypeptides besides the extracellular recognition domain.
  • the multimerization (e.g., dimerization, trimerization, etc.) of an anti-idiotype polypeptide activates proliferative and/or survival signaling.
  • Such embodiments include, for example, fusion polypeptides where the extracellular recognition domain is attached to a lymphoproliferative element, and an antiidiotype polypeptide with the intracellular domains from a lymphoproliferative element, for example an intracellular signaling domain from a cytokine receptor.
  • multimerization of an anti-idiotype polypeptide activates cell death signaling.
  • Such embodiments include, for example, an antiidiotype polypeptide with the intracellular domains from some of the apoptosis-inducing polypeptides disclosed herein.
  • the target antibody or antibody mimetic recognized by an antiidiotype polypeptide includes an Fc domain from IgM or IgA, and binding of the target induces multimerization (e.g., trimerization and higher order multimerization).
  • the target antibody or antibody mimetic can activate intracellular domains that require higher order multimerization (e.g., trimerization).
  • the anti-idiotype polypeptide recognizes nebacumab, which includes an Fc domain from IgM, and binding of the the anti-idiotype polypeptide to nebacumab induces multimerization, and activation of the intracellular domain of the anti-idiotype polypeptide.
  • the intracellular domain can include one or more, two or more, three or more, or all of the domains, motifs, and/or mutations of any of the intracellular domains disclosed herein. In some embodiments, the intracellular domain can include one or more, two or more, three or more, or all of the domains, motifs, and/or mutations of any intracellular domains known to induce proliferation and/or survival of T cells and/or NK cells. In some embodiments, the intracellular domain can activate a Jak pathway, a Stat pathway, a Jak/Stat pathway, a TRAF pathway, a PI3K pathway, and/or a PLC pathway.
  • intracellular domains for activating the various pathways are disclosed in the “Lymphoproliferative elements” herein and can be used as intracellular domain of an anti-idiotype polypeptide.
  • the intracellular domain of an anti-idiotype polypeptide can include all or part of one or more intracellular signaling domains from one or more cytokine receptors.
  • the one or more cytokine receptors can be selected from CD27, CD40, CRLF2, CSF2RA, CSF2RB, CSF3R, EPOR, GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2R, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R, IL6ST, IL7R, IL7RA, IL9R, IL10RA, IL10RB, IL11RA, IL12RB1, IL13R, IL13RA1, IL13RA2, IL15R, IL15RA, IL17RA, IL17RB, IL17RC, IL17RE, IL18R1, IL18RAP, IL20RA, IL20RB, IL21R, IL22RA1, IL23R, IL27
  • the intracellular domain of an anti-idiotype polypeptide includes one or more apoptotic domains known to induce cell death in T cells and/or NK cells. Such intracellular domains are also referred to herein as intracellular apoptotic domains and include domains from apoptosisinducing polypeptides, referred to herein as apoptotic polypeptides, and functional fragments thereof.
  • the intracellular apoptotic domain of an anti-idiotype polypeptide can include one or more caspase activation and recruitment domains (CARDs), death domains (DDs), death effector domains (DEDs), pyrin domains (PYDs), and/or caspase proteolytic domains.
  • such apoptotic polypeptides are capable of inducing an apoptotic signal upon dimerization.
  • a skilled artisan will understand how to identify and incorporate these intracellular apoptotic domains (and other apoptosis-inducing domains) into an antiidiotype polypeptide of the current disclosure.
  • the intracellular apoptotic domain of an anti-idiotype polypeptide can include one or more CARDs, DDs, DEDs, PYDs, and/or caspase proteolytic domains.
  • the intracellular apoptotic domain of an anti-idiotype polypeptide can include one or more means for performing the function of a CARD, DD, DED, PYD, and/or caspase proteolytic domain of a caspase, such as of a caspase 2, 8, 9, or 10.
  • the intracellular apoptotic domain can include one or more CARDs from Apaf-1, DARK, CED-4, CED-3, Drone, CARMA1, Bcl-10, Nodi, Nod2, RIP2, ICEBERG, RIG-I, MDA5, MAV5, ASC, NALP1, caspase 1, caspase 2, caspase-5, and/or caspase 9, and/or functional fragments thereof.
  • the intracellular apoptotic domain can include one or more DDs from TNF-R1, Fas, p75, TRADD, FADD, RIP, MyD88, IRAKs, Pelle, Tube, PIDD, RAIDD, and/or MALT1, and/or functional fragments thereof.
  • the intracellular apoptotic domain can include one or more DEDs from FADD, caspase 8, caspase 10, c-FLIP, v-FLIPs, MC159, PEA-15, DEDD, and/or DEDD2, and/or functional fragments thereof.
  • the intracellular apoptotic domain can include one or more PYDs from ASC, ASC2, NALP1, NALP1, NALP3, NALP4, NALP5, NALP6, NALP7, NALP8, NALP9, NALP10, NALP11, and/or NALP12, and/or functional fragments thereof.
  • the intracellular apoptotic domain can include one or more domains from caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, caspase 14, FAS (CD95 or Apo-1 antigen), TNF-R1, Death Receptor 3 (DR3), Death Receptor 4 (DR4 or TRAIL Receptor-I), Death Receptor 5 (DR5 or TRAIL Receptor-II), FADD, APAF1, CRADD/RAIDD, ASC, Bcl-2 family members, Bax, Bak, RIPK3, and/or RIPK1-RHIM, and/or functional fragments thereof.
  • Bax and Bak are pro-apoptotic Bcl-2 family members that can cause mitochondrial depolarization (or mislocalization of anti-apoptotic family members, like Bcl-xL or Bcl-2).
  • the RIPK3 and RIPK1-RHIM domain can trigger a related form of pro-inflammatory cell death, called necroptosis, due to MLKL-mediated membrane lysis.
  • the intracellular apoptotic domain includes one or more of a caspase 2 polypeptide, a caspase 8 polypeptide, a caspase 9 polypeptide, and/or a caspase 10 polypeptide.
  • Caspase 2, caspase 8, caspase 9, and caspase 10 are also referred to herein as initiator caspases.
  • the intracellular domain of an anti-idiotype polypeptide includes a fusion polypeptide of one or more domains of an initiator caspase and one or more domains of an effector caspase.
  • the initiator caspase can be caspase 2, caspase 8, caspase 9, and/or caspase 10.
  • the effector caspase can be caspase 3, caspase 6, and/or caspase 7.
  • intracellular apoptotic domain of an anti-idiotype polypeptide can include at least one domain from one or more of caspase 2, caspase 8, caspase 9, or caspase 10, or a functional fragment thereof.
  • the functional fragment is an intracellular domain from a caspase polypeptide that lacks one or more domains.
  • the one or more domains lacking in the functional fragment can be CARDs, DDs, DEDs, PYDs, and/or caspase proteolytic domains.
  • the functional fragment is a caspase 2 polypeptide lacking a CARD.
  • the functional fragment is a caspase 8 polypeptide lacking a DED.
  • the intracellular apoptotic domain of an anti-idiotype polypeptide includes one or more domains from a caspase 2 polypeptide.
  • the intracellular apoptotic domain includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of SEQ ID NO:680.
  • the intracellular apoptotic domain includes all domains of the protein of SEQ ID NO:680.
  • the intracellular apoptotic domain includes one or more domains of the polypeptide of SEQ ID NO:680.
  • the intracellular apoptotic domain includes one or more domains of the polypeptide of SEQ ID NO:680.
  • the intracellular apoptotic domain includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the CARD of caspase 2 (amino acids 32 to 118 of SEQ ID NO:680) and/or the CASc domain of caspase 2 (amino acids 192 to 447 of SEQ ID NO:680).
  • the intracellular apoptotic domain includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids of SEQ ID NO:680, wherein the polypeptide sequence does not include a stretch of at least 10, 15, 20, or all the amino acids of the CARD (amino acids 32-118 of SEQ ID NO:680) and/or the CASc domain of caspase 2 (amino acids 192 to 447 of SEQ ID NO:680).
  • the intracellular apoptotic domain includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of amino acids 327 to 452 of SEQ ID NO:680 or of amino acids 359 to 452 of SEQ ID NO:680.
  • the intracellular apoptotic domain of an anti-idiotype polypeptide includes one or more domains from a caspase 8 polypeptide.
  • the intracellular apoptotic domain includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of SEQ ID NO:681. In some embodiments, the intracellular apoptotic domain includes all domains of the protein of SEQ ID NO:681. In some embodiments, the intracellular apoptotic domain includes one or more domains of the protein of SEQ ID NO:681.
  • the intracellular apoptotic domain includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the DED of caspase 8 (amino acids 3 to 84 of SEQ ID NO:681), the DD of caspase 8 (amino acids 134 to 212 of SEQ ID NO:681), and/or CASc domain of caspase 8 amino acids 242 to 494 of SEQ ID NO:681.
  • the intracellular apoptotic domain includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids of SEQ ID NO:681, wherein the polypeptide sequence does not include a stretch of at least 10, 15, 20, or all the amino acids of the DED of caspase 8 (amino acids 3 to 84 of SEQ ID NO:681), the DD of caspase 8 (amino acids 134 to 212 of SEQ ID NO:681), and/or CASc domain of caspase 8 amino acids 242 to 494 of SEQ ID NO:681.
  • the intracellular apoptotic domain includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of amino acids 384 to 496 of SEQ ID NO:681 or of amino acids 411 to 496 SEQ ID NO:681.
  • the intracellular apoptotic domain of an anti-idiotype polypeptide includes one or more domains from a caspase 9 polypeptide.
  • the intracellular apoptotic domain includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids of SEQ ID NO:682.
  • the intracellular apoptotic domain includes all domains of the protein of SEQ ID NO:682.
  • the intracellular apoptotic domain includes one or more domains of the protein of SEQ ID NO:682.
  • the intracellular apoptotic domain includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the CARD of caspase 9 (amino acids 17 to 90 of SEQ ID NO:682) and/or the CASc domain of caspase 9 (amino acids 152 to 414 of SEQ ID NO:682).
  • the intracellular apoptotic domain includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids of SEQ ID NO:682, wherein the polypeptide sequence does not include a stretch of at least 10, 15, 20, or all the amino acids of the CARD of caspase 9 (amino acids 17 to 90 of SEQ ID NO:682) and/or the CASc domain of caspase 9 (amino acids 152 to 414 of SEQ ID NO:682).
  • the intracellular apoptotic domain includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of amino acids 294 to 416 of SEQ ID NO:682 or of amino acids 336 to 416 of SEQ ID NO:682.
  • the intracellular apoptotic domain of an anti-idiotype polypeptide includes one or more domains from a caspase 10 polypeptide.
  • the intracellular apoptotic domain includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of SEQ ID NO:683.
  • the intracellular apoptotic domain includes all domains of the protein of SEQ ID NO:683.
  • the intracellular apoptotic domain includes one or more domains of the protein of SEQ ID NO:683.
  • the intracellular apoptotic domain includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the DD of caspase 10 (amino acids 18 to 99 of SEQ ID NO:683), the DED of caspase 10 (amino acids 112 to 190 of SEQ ID NO:683), and/or the CASc domain of caspase 10 (amino acids 233 to 474 of SEQ ID NO:683).
  • the intracellular apoptotic domain includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids of SEQ ID NO:683, wherein the polypeptide sequence does not include a stretch of at least 10, 15, 20, or all the amino acids of the DD of caspase 10 (amino acids 18 to 99 of SEQ ID NO:683), the DED of caspase 10 (amino acids 112 to 190 of SEQ ID NO:683), and/or the CASc domain of caspase 10 (amino acids 233 to 474 of SEQ ID NO:683).
  • the intracellular apoptotic domain includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of amino acids 365 to 478 of SEQ ID NO:683 or of amino acids 388 to 478 of SEQ ID NO:683.
  • the intracellular apoptotic domain of an anti-idiotype polypeptide can include one or more means for activating an initiator caspase or an effector caspase, or both. In some embodiments, the intracellular apoptotic domain of an anti-idiotype polypeptide can include one or more means for binding to a death domain. In some embodiments, the intracellular apoptotic domain of an antiidiotype polypeptide can include one or more means for performing the function of a death domain of caspase 2, 3, 9, or 10, or FAS or a TNF receptor ICD. In some illustrative embodiments, these antiidiotype polypeptides further comprise a dimerizing moiety, wherein the dimerizing moiety is constitutively dimerized.
  • the intracellular apoptotic domain of an anti-idiotype polypeptide intracellular domain includes one or more domains from a FAS (CD95 or Apo-I antigen) polypeptide.
  • the intracellular apoptotic domain includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of SEQ ID NO:684.
  • the intracellular apoptotic domain includes all domains of the protein of SEQ ID NO:684.
  • the intracellular apoptotic domain includes one or more domains of the protein of SEQ ID NO:684.
  • the intracellular apoptotic domain includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the DD of FAS polypeptide (amino acids 257 to 341 of SEQ ID NO:684).
  • the intracellular apoptotic domain includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids of SEQ ID NO:684, wherein the polypeptide sequence does not include a stretch of at least 10, 15, 20, or all the amino acids of the DD of FAS polypeptide (amino acids 257 to 341 of SEQ ID NO:684).
  • the intracellular apoptotic domain of an anti-idiotype polypeptide includes one or more domains from a TNF-R1 (Tumor necrosis factor receptor superfamily member 1) polypeptide.
  • the intracellular apoptotic domain includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of SEQ ID NO:685.
  • the intracellular apoptotic domain includes all domains of the protein of SEQ ID NO: 685.
  • the intracellular apoptotic domain includes one or more domains of the protein of SEQ ID NO:685. In some embodiments, the intracellular apoptotic domain includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the DD of TNF-R1 polypeptide (amino acids 358 to 438 of SEQ ID NO:685).
  • the intracellular apoptotic domain includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids of SEQ ID NO:685, wherein the polypeptide sequence does not include a stretch of at least 10, 15, 20, or all the amino acids of the DD of TNF-R1 polypeptide (amino acids 358 to 438 of SEQ ID NO:685).
  • the intracellular apoptotic domain of an anti-idiotype polypeptide includes one or more domains from a DR-3 (Tumor necrosis factor receptor superfamily member 25) polypeptide.
  • the intracellular apoptotic domain includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of SEQ ID NO:686.
  • the intracellular apoptotic domain includes all domains of the protein of SEQ ID NO: 686.
  • the intracellular apoptotic domain includes one or more domains of the protein of SEQ ID NO:686.
  • the intracellular apoptotic domain includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the DD of DR-3 polypeptide (amino acids 32 to 145 of SEQ ID NO:686).
  • the intracellular apoptotic domain includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids of SEQ ID NO:686, wherein the polypeptide sequence does not include a stretch of at least 10, 15, 20, or all the amino acids of the DD of DR-3 polypeptide (amino acids 32 to 145 of SEQ ID NO:686).
  • the intracellular apoptotic domain of an anti-idiotype polypeptide includes one or more domains from a DR-4 (Tumor necrosis factor receptor superfamily member 10A; TRAIL Receptor-I) polypeptide.
  • the intracellular apoptotic domain includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of SEQ ID NO:687.
  • the intracellular apoptotic domain includes all domains of the protein of SEQ ID NO:687.
  • the intracellular apoptotic domain includes one or more domains of the protein of SEQ ID NO:687.
  • the intracellular apoptotic domain includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the DD of DR-4 polypeptide (amino acids 367 to 454 of SEQ ID NO:687).
  • the intracellular apoptotic domain includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids of SEQ ID NO:687, wherein the polypeptide sequence does not include a stretch of at least 10, 15, 20, or all the amino acids of the DD of DR-4 polypeptide (amino acids 367 to 454 of SEQ ID NO:687).
  • the intracellular apoptotic domain of an anti-idiotype polypeptide includes one or more domains from a DR-5 (Tumor necrosis factor receptor superfamily member 10B; TRAIL Receptor-II) polypeptide.
  • the intracellular apoptotic domain includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of SEQ ID NO:688.
  • the intracellular apoptotic domain includes all domains of the protein of SEQ ID NO: 688.
  • the intracellular apoptotic domain includes one or more domains of the protein of SEQ ID NO:688.
  • the intracellular apoptotic domain includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the DD of DR-5 polypeptide (amino acids 341 to 428 of SEQ ID NO:688).
  • the intracellular apoptotic domain includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids of SEQ ID NO:688, wherein the polypeptide sequence does not include a stretch of at least 10, 15, 20, or all the amino acids of the DD of DR-5 polypeptide (amino acids 341 to 428 of SEQ ID NO:688).
  • the anti-idiotype polypeptide includes a cleavable signal.
  • the cleavable signal can include a binding-induced proteolytic cleavage site of a Notch receptor.
  • the cleavable signal can include a gamma secretase recognition sequence.
  • the anti-idiotype polypeptide includes a Notch receptor that includes an extracellular domain having a recognition site for a target antibody or a target antibody mimetic, a transmembrane domain having a binding induced proteolytic cleavage site, and one or more intracellular domains.
  • the intracellular domains can include one or more transcription factor.
  • the extracellular domain of the Notch receptor leads to intramembrane proteolysis through dimerization of the receptor (sequential proteolysis by a disintegrin and metalloproteinase (ADAM) metalloprotease and the gamma-secretase complex).
  • ADAM disintegrin and metalloproteinase
  • the induced cleavage of the receptor releases the intracellular fragment of Notch.
  • the notch receptor which is required for dimerization, can be absent from the anti-idiotype polypeptide.
  • the anti-idiotype polypeptide can include an extracellular recognition domain, which can provide dimerization upon addition of the antibody or antibody mimetic.
  • the Notch intracellular domain is a transcriptional regulator that is released from the membrane upon dimerization and translocates into the nucleus to activate target genes that play key roles in cell-cell signaling during development.
  • the intracellular domain of an anti-idiotype polypeptide can include the Notch intracellular domain.
  • intracellular transcriptional domains Such intracellular domains on anti-idiotype polypeptides that regulate transcription are referred to herein as intracellular transcriptional domains, and can increase or decrease expression of target genes to which the intracellular transcriptional domains bind.
  • the intracellular domain is a transcriptional activator and increases expression.
  • the intracellular domain is a transcriptional repressor and decreases expression.
  • the intracellular domain of an anti-idiotype polypeptide can be an artificial transcription factor.
  • the artificial transcription factor can include domains from zinc-fmger nucleases, Gal4, and/or tetR, or any other transcription factor known in the art.
  • the intracellular domain is a site-specific nuclease.
  • the site-specific nuclease can be one or more of zinc finger nuclease (ZFN), Transcription activator-like effector nucleases (TALEN), CRISPR/Cas9 system.
  • the intracellular domain includes one or more of the domains of a Caspase 9 polypeptide.
  • the intracellular domain is a recombinase. In some embodiments, the intracellular domain is an inhibitory immunoreceptor. In some embodiments, the intracellular domain is an activating immunoreceptor. In some embodiments, release of the intracellular domain modulates proliferation of the cell. In some embodiments, release of the intracellular domain modulates apoptosis in the cell. In some embodiments, release of the intracellular domain induces cell death by a mechanism other than apoptosis. In some embodiments, release of the intracellular domain modulates gene expression in the cell through transcriptional regulation, chromatin regulation, translation, trafficking or post-translational processing. In some embodiments, release of the intracellular domain modulates differentiation of the cell.
  • release of the intracellular domain modulates migration of the cell. In some embodiments, release of the intracellular domain modulates the expression and secretion of a molecule from the cell. In some embodiments, release of the intracellular domain modulates adhesion of the cell to a second cell or to an extracellular matrix. In some embodiments, release of the intracellular domain induces de novo expression a gene product in the cell.
  • release of the intracellular domain induces de novo expression a gene product in the cell, wherein the gene product is a transcriptional activator, a transcriptional repressor, a chimeric antigen receptor, a second chimeric Notch receptor polypeptide, a translation regulator, a cytokine, a hormone, a chemokine, or an antibody.
  • the gene product is a transcriptional activator, a transcriptional repressor, a chimeric antigen receptor, a second chimeric Notch receptor polypeptide, a translation regulator, a cytokine, a hormone, a chemokine, or an antibody.
  • the anti-idiotype polypeptide can include a cleavable signal that can be one or more gamma secretase substrate sequences that are recognizable by gamma secretase for cleavage.
  • the gamma secretase substrate sequences are the sequences from the substrates of the gamma- secretase owing to which the substrate gets cleaved.
  • the one or more gamma secretase substrate sequences can be from one or more polypeptides such as ERBB4, INSR, IGF1R, CSF1R, VEGFR1, VEGFR2, VEGFR3, FGFR3, FGFR4, PTK7, TRKA, TRKB, MUSK, MET, AXL, MER, TYRO3, TIE1, EPHA2, EPHA4, EPHA5, EPHA7, EPHB2, EPHB3, EPHB4, EPHB6, RYK, Alcadein a, Alcadein [3, Alcadein y, APLP1, APLP2, APP, CD44, E-Cadherin, EpCAM, GHR, IL-1R2, Neuregulin-1, Notch-1, Notch-2, Notch-3, Notch-4, P75 NTR, Podoplanin, PTK7, and Syndecan-3.
  • polypeptides such as ERBB4, INSR, IGF1R, CSF1R, VEGFR1, VEGFR2, VEGFR3,
  • the anti-idiotype polypeptide can include gamma secretase substrate sequence of Alcadein a polypeptide.
  • the anti-idiotype polypeptide includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all the amino acids of SEQ ID NO:689.
  • the anti-idiotype polypeptide can include gamma secretase substrate sequence of Alcadein P polypeptide.
  • the anti-idiotype polypeptide includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all the amino acids of SEQ ID NO:690.
  • the anti-idiotype polypeptide can include gamma secretase substrate sequence of Alcadein y polypeptide.
  • the anti-idiotype polypeptide includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all the amino acids of SEQ ID NO:691.
  • the anti-idiotype polypeptide can include gamma secretase substrate sequence of APLP1 polypeptide.
  • the anti-idiotype polypeptide includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all the amino acids of SEQ ID NO:692.
  • the antiidiotype polypeptide can include gamma secretase substrate sequence of APLP2 polypeptide.
  • the anti-idiotype polypeptide includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all the amino acids of SEQ ID NO:693.
  • the anti-idiotype polypeptide can include gamma secretase substrate sequence of Notch- 1 polypeptide.
  • the antiidiotype polypeptide includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all the amino acids of SEQ ID NO:694.
  • the anti-idiotype polypeptide can include gamma secretase substrate sequence of Notch-2 polypeptide.
  • the anti-idiotype polypeptide includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all the amino acids of SEQ ID NO:695.
  • the anti-idiotype polypeptide can include gamma secretase substrate sequence of Notch-3 polypeptide.
  • the anti-idiotype polypeptide includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all the amino acids of SEQ ID NO:696.
  • the anti-idiotype polypeptide can include gamma secretase substrate sequence of Notch-4 polypeptide.
  • the anti-idiotype polypeptide includes a polypeptide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all the amino acids of SEQ ID NO:697.
  • the anti-idiotype polypeptide can include a linker between any two adjacent domains.
  • a linker can be between the transmembrane domain and the first intracellular domain.
  • the extracellular recognition domain of the anti-idiotype can be an antibody and a linker can be between the heavy chain and the light chain.
  • a linker can be between the extracellular recognition domain and the transmembrane domain and another linker can be between the transmembrane domain and the intracellular domain.
  • a linker can be between a first intracellular domain and a second intracellular domain.
  • the linker can be between the extracellular recognition domain and the intracellular domain.
  • the linker peptide may have any of a variety of amino acid sequences. Proteins can be joined by a spacer peptide, generally of a flexible nature, although other chemical linkages are not excluded.
  • a linker can be a peptide of between about 1 and about 100 amino acids in length, or between about 1 and about 25 amino acids in length. These linkers can be produced by using synthetic, linker-encoding oligonucleotides to couple the proteins. Peptide linkers with a degree of flexibility can be used.
  • the linking peptides may have virtually any amino acid sequence, bearing in mind that suitable linkers will have a sequence that results in a generally flexible peptide. The use of small amino acids, such as glycine and alanine, are typically of use in creating a flexible peptide. The creation of such sequences is routine to those of skill in the art.
  • Suitable linkers can be readily selected and can be of any of a suitable of different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.
  • Exemplary flexible linkers include glycine polymers (G) n , glycine-serine polymers (including, for example, (GS) n , (GSGGS) n , (GGS) n , (GGGS) n , and (GGGGS) n where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are of interest since both of these amino acids are relatively unstructured, and therefore may serve as a neutral tether between components.
  • Glycine polymers are of particular interest since glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)).
  • Exemplary flexible linkers include, but are not limited GGGGSGGGGS (SEQ ID NO:674), GGGGSGGGGSGGGGS (SEQ ID NO:63), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:372), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:675), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:64), GGSSRSS (SEQ ID NO:673), GGGGSGGGSGGGGS (SEQ ID NO:65), GGSG (SEQ ID NO:66), GGSGG (SEQ ID NO:67), GSGSG (SEQ ID NO:68), GSGGG (SEQ ID NO:69), GGGSG (SEQ ID NO:70), GSSSG (SEQ ID N0:71), and the like.
  • the ordinarily skilled artisan will recognize that design of a peptide conjugated to any elements described above can include linkers that are all or partially flexible, such that the linker can include a flexible linker
  • Anti-idiotype polypeptides can function as single polypeptides, or can function as fusions to other polypeptides.
  • the fusion polypeptides can include only the extracellular recognition domains of one or more anti-idiotype polypeptides or the fusion polypeptides can further include one or more of the other domains of the anti-idiotype polypeptides.
  • the fusion polypeptides can have different activities based on the presence or absence of the target antibody or antibody mimetic.
  • a constitutively active lymphoproliferative element can include an extracellular recognition domain from an anti-idiotype polypeptide that recognizes a target antibody.
  • the constitutively active lymphoproliferative element can promote proliferation and/or survival of the cell expressing the fusion polypeptide.
  • the target antibody for example a target antibody that induces ADCC
  • binding of the target antibody to the extracellular recognition domain can result in death of the cell expressing the fusion polypeptide with the constitutive lymphoproliferative element.
  • the extracellular recognition domain of a fusion polypeptide can perform any of the functions of an anti-idiotype polypeptide disclosed herein, including, for example, acting as a safety switch and acting as an activity modulator.
  • the membrane association domain of the anti-idiotype polypeptide can be provided by the polypeptide to which the anti-idiotype polypeptide is fused.
  • an anti-idiotype polypeptide, or the anti-idiotype extracellular recognition domain of an anti-idiotype polypeptide is expressed as part of a fusion polypeptide.
  • the fusion polypeptide can be a fusion between the anti-idiotype polypeptide or the extracellular recognition domain of an anti-idiotype polypeptide and a first engineered signaling polypeptide, which are disclosed in more detail elsewhere herein.
  • the anti-idiotype polypeptide or the extracellular recognition domain of an anti-idiotype polypeptide is fused to a lymphoproliferative element, CAR, and/or a recombinant TCR.
  • the antiidiotype polypeptide or the extracellular recognition domain of an anti-idiotype polypeptide is fused to a cytokine.
  • the fusion polypeptide can further include one or more of the other domains of the anti-idiotype polypeptides.
  • the fusion polypeptide including an anti-idiotype polypeptide can further include a CAR.
  • the fusion polypeptide can include a recombinant TCR.
  • the extracellular recognition domain of the anti-idiotype polypeptide can be expressed as part of the antigenspecific binding region (ASTR, as discussed elsewhere herein) of the CAR (e.g., a bispecific antibody) or the antigen binding site of the recombinant TCR, or the extracellular recognition domain can be expressed as part of a separate domain on the fusion polypeptide.
  • ASTR antigenspecific binding region
  • the CAR and the extracellular recognition domain from the anti-idiotype polypeptide can each be one part of a bispecific antibody that is attached to the other domains of the fusion polypeptide.
  • an anti-idiotype polypeptide for example a safety switch or an activity modulator, is expressed fused to a lymphoproliferative element, to form a fusion polypeptide.
  • the fusion polypeptide can include a lymphoproliferative element.
  • Such constructs provide the advantage, especially in combination with other “space saving” elements provided herein, of taking up less genomic space on an RNA genome compared to separate polypeptides.
  • the lymphoproliferative element of a fusion polypeptide can be any of the lymphoproliferative elements disclosed elsewhere herein.
  • the lymphoproliferative element of the fusion polypeptide can include one or more or all of the domains, motifs, and/or mutations of the intracellular signaling domains disclosed herein or otherwise known to induce proliferation and/or survival of T cells and/or NK cells.
  • the lymphoproliferative element of a fusion polypeptide is constitutively active.
  • the lymphoproliferative element of a fusion polypeptide is an inducible lymphoproliferative element.
  • binding of the target antibody or antibody mimetic to the extracellular recognition domain on the fusion polypeptide dimerizes two intracellular signaling domains of the inducible lymphoproliferative element to drive proliferation of cells such as T cells and/or NK cells.
  • the inducible lymphoproliferative element of a fusion polypeptide activates proliferative and/or survival signaling after binding of the anti-idiotype extracellular recognition domain to the target antibody or antibody mimetic, as also discussed in the “Activity modulators” section herein.
  • a fusion polypeptide that includes an inducible lymphoproliferative element can include a dimerization domain besides the anti-idiotype extracellular recognition domain.
  • a fusion polypeptide that includes an inducible lymphoproliferative element does not include a dimerization domain besides the anti-idiotype extracellular recognition domain.
  • an eTag is expressed as a fusion polypeptide, fused the 5’ terminus of the c-Jun domain (SEQ ID NO: 104), a transmembrane domain from CSF2RA (SEQ ID NO: 129), a first intracellular domain from MPL (SEQ ID NO:283), and a second intracellular domain from CD40 (SEQ ID NO:208).
  • the cell tag may be associated with the cell membrane via its natural membrane attachment sequence or via a heterologous membrane attachment sequence such as a GPI- anchor or transmembrane sequence.
  • cell tags are expressed on the T cell and/or NK cell but are not expressed on the replication incompetent recombinant retroviral particles.
  • polynucleotides, polypeptides, and cells comprise 2 or more safety switches.
  • At least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% of the neutrophils, basophils, and/or eosinophils present in a blood sample that is subjected to a method for modifying herein, are present in the cell formulation, including at the time of the optional delivery (i.e., administering) step.
  • At least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% of the B cells present in a blood sample that is subjected to a method for modifying herein, are present in the cell formulation, including at the time of the optional delivery step.
  • At least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% of the monocytes present in a blood sample that is subjected to a method for modifying herein, are present in the cell formulation, including at the time of the optional delivery step.
  • the volume of the cell formulation including the modified lymphocytes is less than traditional CAR-T methods, which typically are infusion-delivery methods, and can be less than, or less than about 1 ml, about 2 ml, about 3 ml, about 4 ml, about 5 ml, about 10 ml, about 15 ml, about 20 ml, or about 25 ml.
  • the short contacting time in certain embodiments results in many of the modified lymphocytes in cell formulations herein, having on their surfaces, binding polypeptides, fusogenic polypeptides, and in some embodiments T cell activation elements that originated on the surface of retroviral particles, either through association with the recombinant retroviral particles or by fusion of the retroviral envelopes with the plasma membranes, including at the time of the optional delivery step.
  • the modified lymphocytes in the cell formulation include a pseudotyping element and/or a T cell activation element, e.g., a T cell activating antibody.
  • the pseudotyping element and/or T cell activation element can be bound to the surface of the modified lymphocytes through, for example, a T cell receptor, CD28, 0X40, 4-1BB, ICOS, CD9, CD53, CD63, CD81, CD82, and/or the pseudotyping element and/or T cell activation element can be present in the plasma membrane of the modified lymphocytes.
  • Cell formulations are provided herein, that include for example T cells and/or NK cells. Such formulations, in illustrative embodiments are provided by methods provided herein. Any of the cell formulations provided herein can include self-driving CAR-T cells. In one aspect, provided herein is a cell formulation comprising a population of self-driving CAR-T cells, such as modified, genetically modified, transcribed, transfected, and/or stably integrated self-driving CAR-T cells in a delivery solution.
  • lymphocytes are contacted with recombinant nucleic acid vectors and modified lymphocytes are ex vivo after such contacting in some illustrative embodiments provided herein, in these embodiments some or all of the T and NK cells do not yet express the recombinant nucleic acid or have not yet integrated the recombinant nucleic acid into the genome of the cell, and some of the retroviral particles in embodiments including these, may be associated with, but may have not fused with the target cell membrane, before being used or included in any of the methods or compositions provided herein, including, but not limited to, being introduced or reintroduced back into a subject, or before being used to prepare a cell formulation.
  • cell formulation aspects and embodiments are provided herein that can be produced, for example, from these illustrative methods provided herein, such as for example, rapid point of care methods that in illustrative embodiments involve subcutaneous administration.
  • Such cell formulations including but not limited to those set out immediately below and in the Exemplary Embodiments section herein, can exist at the time of collection of cells after they are contacted with a recombinant retroviral vector and optionally rinsed, and can exist up to and including at the time of administration to a subject, in illustrative embodiments subcutaneously.
  • cell formulations comprising T cells and/or NK cells, wherein less than 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 10%, or 5% of the cells in the cell formulation are T cells and/or NK cells.
  • cell formulations comprising lymphocytes, NK cells, and/or T cells, are provided wherein at least 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the lymphocytes, NK cells, and/or in illustrative embodiments T cells in the cell formulation are modified cells, for example, modified with polynucleotides comprising nucleic acids that encode anti-idiotype polypeptides provided herein. Such polynucleotides can optionally encode a CAR, TCR, inhibitory RNA, or LE, as provided herein.
  • lymphocytes between 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, and 70% of the lymphocytes are modified on the low end of the range and 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95% of the lymphocytes are modified cells on the high end of the range, for example between 5% and 95%, 10% and 90%, 25% and 75%, and 25% and 95%.
  • At least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or all of the modified lymphocytes within the cell formulation are not genetically modified, transduced, or stably transfected.
  • the modified lymphocytes are not genetically modified, transduced, or stably transfected on the low end of the range and 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% or all of the modified lymphocytes are not genetically modified, transduced, or stably transfected on the high end of the range, for example between 5% and 95%, 10% and 90%, 25% and 75%, and 25% and 95%.
  • the polynucleotide of genetically modified lymphocytes can be either extrachromosomal or integrated into the genome in these cell formulations that are formed after contacting and incubation, and at the time of optional administration. In some embodiments of these cell formulations, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or all of the genetically modified lymphocytes have an extrachromosomal polynucleotide.
  • between 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, and 70% of the modified or genetically modified lymphocytes have an extrachromosomal polynucleotide on the low end of the range and 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% or all of the modified or genetically modified lymphocytes have an extrachromosomal polynucleotide on the high end of the range, for example between 5% and 95%, 10% and 90%, 25% and 75%, and 25% and 95%.
  • At least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or all of the modified or genetically modified lymphocytes are not transduced or stably transfected in these cell formulations, for example as a result of methods for genetically modifying T cells and/or NK cells provided herein.
  • the modified or genetically modified lymphocytes are not transduced on the low end of the range and 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% or all of the modified or genetically modified lymphocytes are not transduced or stably transfected on the high end of the range, for example between 5% and 95%, 10% and 90%, 25% and 75%, and 25% and 95%.
  • T cells and/or NK cells are contacted with retroviral particles to modify the T cells and/or N cells within hours of delivery, some or most of the reverse transcriptase and integrase present within the retroviral particles that moves into a T cell and/or NK cell after it fuses with a retroviral particle, would still be present in the modified T cells and/or NK cells at the time of delivery.
  • the volume of cell formulation or other solution administered varies depending on the route of administration, as provided elsewhere herein.
  • Cell formulations injected subcutaneously or intramuscularly typically have smaller volumes than those delivered via infusion.
  • the volume of the cell formulation or other solution including a suspension of the modified, and in illustrative embodiments genetically modified lymphocytes is not more than 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 10 ml, 15 ml, 20 ml, 25 ml, 30 ml, 35 ml, 40 ml, 45 ml, or 50 ml.
  • the volume of the cell formulation or other solution including a suspension of the modified lymphocytes can be between 0.20 ml, 0.25 ml, 0.5 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 10 ml, 15 ml, 20 ml, or 25 ml on the low end of the range and 0.5 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 10 ml, 15 ml, 20 ml, 25 ml, 30 ml, 35 ml, 40 ml, 45 ml, or 50 ml., 30 ml, 35 ml, 40 ml, 45 ml, 50 ml, 75 ml, 100 ml, 125 ml, 250 ml, 500 ml, or 1000 ml on the high end of the range.
  • the volume can be between 0.2 ml and 10 ml, 0.5 ml and 10 ml, 0.5 and 2 ml, 1 ml and 250 ml, 1 ml and 100 ml, 10 ml and 100 ml, or 1 ml and 10 ml. In certain illustrative embodiments, less than 10 ml, between 1 ml and 25 ml, and in illustrative embodiments between 1 ml and 3 ml, between 1 ml and 5 ml, or between 1 ml and 10 ml of a cell formulation that includes modified lymphocytes in delivery solution are administered subcutaneously or intramuscularly.
  • the volume of the solution including the modified lymphocytes can be between 0.20 ml, 0.25 ml, 0.5 ml, 1 ml, 2 ml, 3 ml, 4 ml, and 5 ml on the low end of the range and 0.5 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 10 ml, 15 ml, 20 ml, 25 ml, 30 ml, 35 ml, 40 ml, 45 ml, and 50 ml on the high end of the range.
  • a 70 kg subject is dosed at 1.0 x 10 6 T cells/kg by administering 1 ml of a delivery formulation of T cells at 7.0 x 10 7 cells/ml subcutaneously.
  • the solution can include hyaluronidase when the volume of the solution is at least 2 ml, 3 ml, 4 ml, 5 ml, 10 ml, 15 ml, 20 ml, or 25 ml.
  • the delivery solution can be used to resuspend and/or elute cells from the filter in volumes that can be those provided above.
  • a delivery solution provided herein is an elution solution.
  • modified and in illustrative embodiments genetically modified lymphocytes are introduced or reintroduced into the subject by intradermal, intratumoral or intramuscular administration and in illustrative embodiments, subcutaneous administration using a cell formulation present in a subcutaneous delivery device, such as a sterile syringe that is adapted to deliver a solution subcutaneously.
  • a subcutaneous delivery device is used that holds a solution (e.g., a cell formulation herein) and has an open or openable end, which in illustrative embodiments is the open end of a needle, for administrating the solution (e.g., cell formulation) subcutaneously from the liquid holding portion of the device.
  • Such subcutaneous delivery device is effective for, and in illustrative embodiments adapted for subcutaneous delivery, or effective to inject subcutaneously or adapted to inject subcutaneously.
  • subcutaneous delivery devices that are adapted to deliver a solution subcutaneously include subcutaneous catheters, such as indwelling subcutaneous catheters, such as for example, the Insuflon® (Becton Dickinson) and needless closed indwelling subcutaneous catheter systems, for example with wings, such as for example, the Saf-T-Intima® (Becton Dickinson).
  • the delivery device can include a pump, for example an infusion pump or a peristaltic pump.
  • the cell formulation is fluidly connected to any of the needles disclosed herein, for example a needle compatible with, effective for, adapted for, or adapted to deliver subcutaneously or effective to deliver subcutaneously
  • the delivery solution, a composition in the kit, or the cell formulation includes one or more cytokines such as IL-2, IL-7, IL-15, or IL-21, IL-21 and/or cytokine receptor agonists, such as an IL- 15 agonist.
  • the cytokine does not bind to a cytokine receptor included in the delivery solution, kit, or cell formulation; and/or does not bind to a cytokine receptor that is encoded by a polynucleotide in the delivery solution, cell formulation, or kit.
  • the cytokines can be modified cytokines that, not to be limited by theory, selectively activate complexes that drive proliferation.
  • the modified cytokine is a modified IL-2, for example, a fusion protein with a circularly-permuted IL-2 with the extracellular domain of IL-2Ra (see, e.g., Lopes et al, J Immunother Cancer 2020 Apr; 8(1): e000673).
  • the cytokines, modified cytokines, or cytokine receptor agonists can also be administered in one or administrations separate from the cell formulation, before, contemporaneous to, or after the administration including the delivery solution or cell formulation.
  • two or more separate administrations can be in escalating doses.
  • two or more administrations can be at the same dose.
  • two or more administrations can include the same or different cytokines, modified cytokines, and or cytokine receptor agonists.
  • the separate administrations can be a series of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 administrations. In some embodiments, the separate administrations occur on consecutive days.
  • cells in a first cell mixture are modified with a recombinant nucleic acid vector encoding a target antigen, which can be referred to herein as “artificial antigen presenting cells” or “aAPCs”, and cells in a separate second cell mixture from the same subject are modified to express the CAR that binds the antigen.
  • aAPC artificial antigen presenting cells
  • T-APC T cell
  • modified T-APCs can include, as non-limiting examples, B cells, dendritic cells, and macrophages, and in illustrative embodiments dendritic cells and macrophages such as where a corresponding CAR-T target is a B cell cancer target, and can be generated using methods provided herein where reaction mixtures for modification (e.g., transduction) include a T cell binding polypeptide, such as a polypeptide directed to CD3.
  • the cell mixture is whole blood, isolated TNCs, isolated PBMCs.
  • the first cell mixture can be modified with a recombinant nucleic acid vector encoding a fusion protein of the extracellular domain of Her2 and the transmembrane domain of PDGF and the second cell mixture can be modified with a recombinant nucleic acid vector encoding a CAR directed to HER2.
  • the cells can then be formulated into the delivery solution or otherwise administered to the subject at varying CAR effector cell-to-tar get-cell ratios.
  • the effector-to-target ratio at the time of formulation or administration is, or is about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2;1, about 1:1, about 1:2, about 1:3, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10.
  • target cells are co-administered with the modified T and/or NK cells subcutaneously or intramuscularly.
  • the delivery solution or the cell formulation includes synthetic RNA.
  • the synthetic RNA includes inhibitory RNAs such as siRNAs directed to one or more targets.
  • the targets for these inhibitory RNAs can be any of the targets for siRNAs or miRNAs disclosed elsewhere herein.
  • the synthetic RNA includes mRNA encoding for one or more proteins or peptides.
  • the mRNA encodes for one or more CARs.
  • the CARs may be any CAR composition disclosed herein, including bispecific CARs that include an anti-id ERD as disclosed herein.
  • the mRNA can encode any anti-idiotype polypeptide disclosed herein.
  • the mRNA encodes for the target antibody to an anti-idiotype polypeptide disclosed herein.
  • Such embodiment can have an advantage of providing a target antibody that can be cytotoxic when delivered in soluble form, but less or not cytotoxic when taken up by cells after in vivo administration of an mRNA encoding the target antibody.
  • cells that take up and express the target antibody can become artificial antigen presenting cells for embodiments where the anti-idiotype polypeptide has an ICD that is the ICD of an LE, or of a CAR (e.g. a bi-speficic CAR).
  • the mRNA encodes for one or more cytokines. In some embodiments, mRNA encodes for IL-2 or a functional variant thereof. In some embodiments, the mRNA encodes for IL-7 or a functional variant thereof. In some embodiments, the mRNA encodes for IL- 15 or a functional variant thereof. In some embodiments, the mRNA encodes for IL-21 or a functional variant thereof. In some embodiments, the mRNA encodes one or more proteins or polypeptides that bind and activate a CAR. In some embodiments, the mRNA encodes for an antigen recognized by the ASTR of the CAR. In some embodiments, the mRNA encodes for HER2 or an extracellular domain of HER2.
  • the mRNA encodes for EGFR or an extracellular domain of EGFR. In some embodiments, the mRNA encodes for Axl or an extracellular domain of Axl. In some embodiments, the mRNA encodes for CD19 or an extracellular domain of CD19. In some embodiments, the mRNA encodes for CD22 or an extracellular domain of CD22. In some embodiments, the mRNA encodes for an antibody recognized by the ASTR of the CAR. In some embodiments, the MRNA encoding for an antibody recognized by the ASTR of the CAR is an anti-idiotype antibody directed to the antibody or scFv of the ASTR.
  • the mRNA encodes for an antibody that binds an epitope tag of the CAR and can cross-link two CARs as described elsewhere herein.
  • the mRNA encodes for one or more T and/or NK cell co-stimulatory proteins.
  • co-stimulatory proteins may comprise one or more ligands or antibodies to a co-stimulatory receptor on T and/or NK cells.
  • the co-stimulatory receptor is CD28.
  • the co-stimulatory receptor is 4-1BB.
  • the mRNA encodes a protein or polypeptide that is soluble.
  • the mRNA encodes a protein or polypeptide that is membrane-bound.
  • the membrane-bound protein or polypeptide is operatively linked to a transmembrane domain.
  • the synthetic RNA includes both inhibitory RNAs such as siRNAs directed to one or more targets and mRNA encoding for one or more proteins or peptides.
  • a method for generating mRNA for use in the delivery solution or cell formulation may involve in vitro transcription of a template with specially designed primers, followed by PolyA addition, to produce a construct containing 3’ and 5’ untranslated sequence, a 5’ cap and/or IRES, the nucleic acid to be expressed, and a polyA tail, typically 50-200 bases in length.
  • the synthetic RNA is a naturally occurring, endogenous RNA for the nucleic acid of interest.
  • the RNA is not the naturally occurring, endogenous RNA for the nucleic acid of interest.
  • the RNA is modified to change the stability and/or translation efficiency of the RNA.
  • the 5’ UTR, 3’UTR, Kozak sequence, polyA tail is modified.
  • the RNA includes a 5’ cap.
  • the RNA is encapsulated in lipid-based carrier vehicles.
  • One approach for assembling lipid nanocarriers includes directly mixing of a solution of lipids in ethanol with an aqueous solution of the nucleic acid to obtain lipid nanoparticles (LNPs).
  • the LNPs comprise PEG-conjugated lipid. PEG conjugated lipids prevent the aggregation during particle formation and allow the controlled manufacturing of particles with defined diameters in the range between approximately 50 nm and 150 nm.
  • the LNPs do not comprise PEG.
  • the LNPs comprise poly (glycerol) (PGs), poly(oxazolines), sugar- based systems, and poly (peptides).
  • the polypeptides include polysarcosine (pSAR).
  • the LNPs comprise a dendritic cell targeting moiety.
  • the dendritic cell targeting moiety comprises mannose.
  • the RNA can be added to a cell formulation comprising, or coadministered with, modified and/or genetically modified T cells and/or NK cells in cell formulations and methods provided herein.
  • the RNA is added to the isolated blood of a subject and processed in parallel with the T cells and/or NK cells.
  • the RNA can be formulated separately from the modified and/or genetically modified T cells and/or NK cells.
  • the synthetic RNA may be delivered by any means known in the art for therapeutic delivery of RNA.
  • the RNA is delivered intravenously.
  • the RNA is delivered intraperitoneally.
  • the RNA is delivered intramuscularly.
  • the RNA is delivered intratumorally. In some embodiments, the RNA is delivered intradermally. In illustrative embodiments, the RNA is delivered subcutaneously. In some embodiments, the RNA is delivered at the same site as the site of administration of the modified and/or genetically modified T cells and/or NK cells. In some embodiments, the RNA is delivered at a site adjacent to the site of administration of the modified and/or genetically modified T cells and/or NK cells. In some embodiments, the RNA is administered once. In some embodiments, the RNA is administered, 2, 3, 4, 5, 6 or more times.
  • Recombinant retroviral particles are disclosed in methods and compositions provided herein, for example, to modify cells, as non-limiting examples human cells, primary cells, T cells and/or NK cells to make genetically modified and/or transduced cells, human cells, primary cells, T cells and/or NK cells.
  • the recombinant retroviral particles are themselves aspects of the present invention.
  • the recombinant retroviral particles included in aspects provided herein are replication incompetent, meaning that a recombinant retroviral particle cannot replicate once it leaves the packaging cell.
  • retroviral particles are replication incompetent, and if such retroviral particles include nucleic acids in their genome that are not native to the retrovirus, they are “recombinant retroviral particles.” In illustrative embodiments, the recombinant retroviral particles are lentiviral particles.
  • replication incompetent recombinant retroviral particles for use in transducing cells, typically lymphocytes and illustrative embodiments T cells and/or NK cells.
  • the replication incompetent recombinant retroviral particles can include an envelope protein.
  • the envelope protein can be a pseudotyping element.
  • the envelope protein can be an activation element.
  • the replication incompetent recombinant retroviral particles include both a pseudotyping element and an activation element.
  • the replication incompetent recombinant retroviral particles can include any of the pseudotyping elements discussed elsewhere herein.
  • the replication incompetent recombinant retroviral particles can include any of the activation elements discussed elsewhere herein.
  • a replication incompetent recombinant retroviral particle that includes a polynucleotide with nucleic acids that encode an anti-idiotype polypeptide provided in any of the aspects and embodiments herein.
  • Such polypeptide typically includes nucleic acids that further encode at least one of a CAR, and LE, an inhibitory RNA, and a cytokine.
  • the RIP includes a polynucleotide including: A.
  • the T cell activation element can be any of the activation elements discussed elsewhere herein.
  • the T cell activation element can be anti-CD3 scFvFc.
  • a RIP including a polynucleotide including one or more transcriptional units operatively linked to a promoter active in T cells and/or NK cells, wherein the one or more transcriptional units encode an antiidiotype polypeptide and a first signaling polypeptide including an engineered T cell receptor or a chimeric antigen receptor (CAR) and a second signaling polypeptide including a lymphoproliferative element.
  • the lymphoproliferative element can be a chimeric lymphoproliferative element.
  • the anti-idiotype polypeptide, engineered T cell receptor, CAR, or other transgene is expressed, displayed, and/or otherwise incorporated in the surface of the replication incompetent retroviral particle at a reduced level that is less than 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% of the surface expression compared to when the transgene is expressed from an EFl-a or PGK promoter, and in illustrative embodiments, when the transgene is expressed from an EFl-a or PGK promoter in the absence of additional elements (such as degrons or inhibitory RNAs) to reduce such surface expression.
  • additional elements such as degrons or inhibitory RNAs
  • the gene vector is substantially free of the protein transcript encoded by nucleic acid of the gene vector, and/or the RIPs do not express or comprise a detectable amount of the engineered T cell receptor or CAR on their surface, or express or comprise a reduced amount of the engineered T cell receptor or CAR on their surface.
  • nucleic acid sequences that are included in RIPs are provided throughout this disclosure, such as, for example, pseudotyping elements, activation elements, and membrane bound cytokines, as well as nucleic acid sequences that are included in a genome of a replication incompetent, recombinant retroviral particle such as, but not limited to, nucleic acid sequences encoding an antiidiotype polypeptide, nucleic acid sequences encoding a CAR; nucleic acid sequences encoding a lymphoproliferative element; nucleic acids encoding a cytokine; nucleic acid sequences encoding a control element, such as a riboswitch; a promoter, especially a promoter that is constitutively active or inducible in a T cell; and nucleic acids encoding an inhibitory RNA molecule.
  • various aspects provided herein such as methods of making recombinant retroviral particles, methods for performing adoptive cell therapy, and methods for transducing T cells, produce and/or include replication incompetent, recombinant retroviral particles.
  • Replication incompetent recombinant retroviruses that are produced and/or included in such methods themselves form separate aspects of the present invention as replication incompetent, recombinant retroviral particle compositions, which can be in an isolated form.
  • Such compositions can be in dried down (e.g., lyophilized) form or can be in a suitable solution or medium known in the art for storage and use of retroviral particles.
  • Retroviral particle embodiments herein include those wherein the retroviral particle comprises a genome that includes one or more nucleic acids encoding an anti-idiotype polypeptide and one or more inhibitory RNA molecules.
  • nucleic acids that encode inhibitory RNA molecules that can be included in a genome of a retroviral particle including combinations of such nucleic acids with other nucleic acids that encode a CAR or a lymphoproliferative element other than an inhibitory RNA molecule, are included for example, in the inhibitory RNA section provided herein, as well as in various other paragraphs that combine these embodiments.
  • nucleic acids that are disclosed within packaging cell line aspects disclosed herein.
  • disclosure in this section of a recombinant retroviral particle that includes a genome that encodes one or more (e.g., two or more) inhibitory RNA molecules can be combined with various alternatives for such nucleic acids encoding inhibitory RNA molecules provided in other sections herein.
  • nucleic acids encoding one or more inhibitory RNA molecules can be combined with various other functional nucleic acid elements provided herein, as for example, disclosed in the section herein that focuses on inhibitory RNA molecules and nucleic acid encoding these molecules.
  • RNA molecules provided herein in other sections can be used in recombinant retroviral particle aspects of the present disclosure.
  • Necessary elements of recombinant retroviral vectors such as lentiviral vectors, are known in the art. These elements are included in the packaging cell line section and in details for making replication incompetent, recombinant retroviral particles provided in the Examples section and as illustrated in WO2019/055946.
  • lentiviral particles typically include packaging elements REV, GAG and POL, which can be delivered to packaging cell lines via one or more packaging plasmids, a pseudotyping element, various examples which are provided herein, which can be delivered to a packaging cell line via a pseudotyping plasmid, and a genome, which is produced by a polynucleotide that is delivered to a host cell via a transfer plasmid.
  • This polynucleotide typically includes the viral LTRs and a psi packaging signal.
  • the 5’ LTR can be a chimeric 5’ LTR fused to a heterologous promoter, which includes 5’ LTRs that are not dependent on Tat transactivation.
  • Vpu such as a polypeptide comprising Vpu (sometimes called a “Vpu polypeptide” herein) including but not limited to, Src-FLAG-Vpu
  • Vpu polypeptide including but not limited to, Src-FLAG-Vpu
  • Vpx such as Src-FLAG-Vpx, is packaged within the retroviral particle.
  • Vpu and Vpx is packaged within the retroviral particle for any composition or method aspect and embodiment that includes a retroviral particle provided herein.
  • Retroviral particles included in various aspects of the present invention are in illustrative embodiments, replication incompetent, especially for safety reasons for embodiments that include introducing cells transduced with such retroviral particles into a subject.
  • replication incompetent retroviral particles are used to transduce a cell, retroviral particles are not produced from the transduced cell.
  • Modifications to the retroviral genome are known in the art to assure that retroviral particles that include the genome are replication incompetent.
  • replication competent recombinant retroviral particles can be used.
  • expression vectors can be delivered to packaging cells and/or to T cells using different types of vectors, such as expression vectors.
  • Illustrative aspects of the invention utilize retroviral vectors, and in some particularly illustrative embodiments lentiviral vectors.
  • Other suitable expression vectors can be used to achieve certain embodiments herein.
  • Such expression vectors include, but are not limited to, viral vectors (e.g.
  • viral vectors based on vaccinia virus; poliovirus; adenovirus see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther
  • a retroviral vector e.g.
  • replication incompetent recombinant retroviral particles are a common tool for gene delivery (Miller, Nature (1992) 357:455-460).
  • the ability of replication incompetent recombinant retroviral particles to deliver an unrearranged nucleic acid sequence into a broad range of rodent, primate and human somatic cells makes replication incompetent recombinant retroviral particles well suited for transferring genes to a cell.
  • the replication incompetent recombinant retroviral particles can be derived from the Alpharetrovirus genus, the Betaretrovirus genus, the Gammaretrovirus genus, the Deltaretrovirus genus, the Epsilonretrovirus genus, the Lentivirus genus, or the Spumavirus genus.
  • retroviruses suitable for use in the methods disclosed herein.
  • murine leukemia virus MMV
  • human immunodeficiency virus HIV
  • equine infectious anaemia virus EIAV
  • mouse mammary tumor virus MMTV
  • Rous sarcoma virus RSV
  • Fujinami sarcoma virus FuSV
  • Moloney murine leukemia virus Mo-MLV
  • FBR MSV FBR murine osteosarcoma virus
  • Mo-MSV Abelson murine leukemia virus
  • A-MLV Avian myelocytomatosis virus-29
  • AEV Avian erythroblastosis virus
  • retroviruses A detailed list of retroviruses may be found in Coffin et al (“Retroviruses” 1997 Cold Spring Harbor Laboratory Press Eds: J M Coffin, S M Hughes, H E Varmus pp 758-763). Details on the genomic structure of some retroviruses may be found in the art. By way of example, details on HIV may be found from the NCBI Genbank (i.e., Genome Accession No. AF033819).
  • the replication incompetent recombinant retroviral particles can be derived from the Lentivirus genus. In some embodiments, the replication incompetent recombinant retroviral particles can be derived from HIV, SIV, or FIV. In further illustrative embodiments, the replication incompetent recombinant retroviral particles can be derived from the human immunodeficiency virus (HIV) in the Lentivirus genus. Lentiviruses are complex retroviruses which, in addition to the common retroviral genes gag, pol and env, contain other genes with regulatory or structural function.
  • HIV human immunodeficiency virus
  • a typical lentivirus is the human immunodeficiency virus (HIV), the etiologic agent of AIDS, in vivo, HIV can infect terminally differentiated cells that rarely divide, such as lymphocytes and macrophages.
  • HIV human immunodeficiency virus
  • replication incompetent recombinant retroviral particles provided herein contain Vpx polypeptide.
  • replication incompetent recombinant retroviral particles provided herein comprise and/or contain Vpu polypeptide.
  • a retroviral particle is a lentiviral particle.
  • Such retroviral particle typically includes a retroviral genome within a capsid which is located within a viral envelope.
  • DNA-containing viral particles are utilized instead of recombinant retroviral particles.
  • viral particles can be adenoviruses, adeno-associated viruses, herpesviruses, cytomegaloviruses, poxviruses, avipox viruses, influenza viruses, vesicular stomatitis virus (VSV), or Sindbis virus.
  • VSV vesicular stomatitis virus
  • a skilled artisan will appreciate how to modify the methods disclosed herein for use with different viruses and retroviruses, or retroviral particles.
  • functional units can be included in such genomes to induce integration of all or a portion of the DNA genome of the viral particle into the genome of a T cell transduced with such virus.
  • the HIV RREs and the polynucleotide region encoding HIV Rev can be replaced with N-terminal RGG box RNA binding motifs and a polynucleotide region encoding ICP27.
  • the polynucleotide region encoding HIV Rev can be replaced with one or more polynucleotide regions encoding adenovirus E1B 55-kDa and E4 Orf6.
  • replication incompetent recombinant retroviral particles can include nucleic acids encoding a self-driving CAR, as disclosed elsewhere herein.
  • retroviral particles whose genome comprises one or more first transcriptional units operably linked to an inducible promoter inducible in at least one of a T cell or an NK cell, and one or more second transcriptional units operably linked to a constitutive T cell or NK cell promoter, wherein the number of nucleotides between the 5’ end of the one or more first transcriptional units and the 5’ end of the one or more second transcriptional units is less than the number of nucleotides between the 3’ end of the one or more first transcriptional units and the 3’ end of the one or more second transcriptional units, a.
  • At least one of the one or more first transcriptional units encodes a lymphoproliferative element, b. and wherein at least one of the one or more second transcriptional units encodes a first chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activating domain, and wherein nucleic acids within the first transcriptional unit or the second transcriptional unit encode an anti-idiotype polypeptide according to an of the embodiments provided herein.
  • CAR chimeric antigen receptor
  • the replication incompetent recombinant retroviral particles can further display a T cell activation element.
  • T cells contacted and transduced with these replication incompetent recombinant retroviral particles that include nucleic acids encoding a self-driving CAR can receive an initial boost of transcription from the CAR-stimulated inducible promoters as the T cell activation element can stimulate the inducing signal of the CAR-stimulated inducible promoters.
  • the binding of the T cell activation element can induce the calcium ion influx that results in dephosphorylation of NF AT and its subsequent nuclear translocation and binding to NFAT-responsive promoters.
  • the lymphoproliferative elements transcribed and translated from these CAR- stimulated inducible promoters can give an initial increase in proliferation to these cells.
  • the T cell activation element can be a membrane-bound anti-CD3 antibody, and can be GPI-linked or otherwise displayed on virus.
  • the membrane-bound anti-CD3 antibody can be fused to a viral envelope protein, such as MuLV, VSV-G, a Henipavirus-G such as NiV-G, or variants and fragments thereof.
  • the isolated replication incompetent retroviral particles are a large-scale batch contained in a large-scale container.
  • Such large-scale batch can have titers, for example of 10 6 - 10 8 TU/ml and a total batch size of between IxlO 10 TU and IxlO 13 TU, IxlO 11 TU and IxlO 13 TU, IxlO 12 TU and IxlO 13 TU, IxlO 10 TU and 5xl0 12 TU, or IxlO 11 TU and 5xl0 12 TU.
  • retroviral particles for any aspect or embodiment provided herein are substantially pure, as discussed in more detail herein.
  • the recombinant retroviral genomes in nonlimiting illustrative examples, lentiviral genomes, have a limitation to the number of polynucleotides that can be packaged into the viral particle.
  • the polypeptides encoded by the polynucleotide encoding region can be truncations or other deletions that retain a functional activity such that the polynucleotide encoding region is encoded by less nucleotides than the polynucleotide encoding region for the wild-type polypeptide.
  • the polypeptides encoded by the polynucleotide encoding region can be fusion polypeptides that can be expressed from one promoter.
  • the fusion polypeptide can have a cleavage signal to generate two or more functional polypeptides from one fusion polypeptide and one promoter.
  • some functions that are not required after initial ex vivo transduction are not included in the retroviral genome, but rather are present on the surface of the replication incompetent recombinant retroviral particles via the packaging cell membrane. These various strategies are used herein to maximize the functional elements that are packaged within the replication incompetent recombinant retroviral particles.
  • the recombinant retroviral genome to be packaged can be between 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, and 8,000 nucleotides on the low end of the range and 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, and 11,000 nucleotides on the high end of the range.
  • the retroviral genome to be packaged includes one or more polynucleotide regions encoding a first and second engineering signaling polypeptide as disclosed in detail herein.
  • the recombinant retroviral genome to be packaged can be less than 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, or 11,000 nucleotides.
  • Functions discussed elsewhere herein that can be packaged include required retroviral sequences for retroviral assembly and packaging, such as a retroviral rev, gag, and pol coding regions, as well as a 5' LTR and a 3' LTR, or an active truncated fragment thereof, a nucleic acid sequence encoding a retroviral cis-acting RNA packaging element, and a cPPT/CTS element.
  • required retroviral sequences for retroviral assembly and packaging such as a retroviral rev, gag, and pol coding regions, as well as a 5' LTR and a 3' LTR, or an active truncated fragment thereof, a nucleic acid sequence encoding a retroviral cis-acting RNA packaging element, and a cPPT/CTS element.
  • a replication incompetent recombinant retroviral particle herein can include any one or more or all of the following, in some embodiments in reverse orientation with respect to a 5’ to 3’ orientation established by the retroviral 5’ LTR and 3’ LTR (as illustrated in WO20 19/055946 as a non-limiting example): one or more polynucleotide regions encoding a first and second engineering signaling polypeptide, at least one of which includes at least one lymphoproliferative element; a second engineered signaling polypeptide that can include a chimeric antigen receptor; an miRNA, a control element, such as a riboswitch, which typically regulates expression of the first and/or the second engineering signaling polypeptide; a safety switch polypeptide, an intron, a promoter that is active in a target cell, such as a T cell, a 2A cleavage signal and/or an IRES.
  • a delivery composition or suspension for example for treating or preventing a disease, for example cancer or tumor growth, comprising polynucleotides, such as polynucleotide vectors, in illustrative replication incompetent recombinant retroviral particle (RIPs), or modified cells, such as a modified lymphocytes, modified TILs, modified lymphocytes other than B cells, or modified T cells and/or modified NK cells as an active ingredient.
  • polynucleotides such as polynucleotide vectors, in illustrative replication incompetent recombinant retroviral particle (RIPs), or modified cells, such as a modified lymphocytes, modified TILs, modified lymphocytes other than B cells, or modified T cells and/or modified NK cells as an active ingredient.
  • RIPs replication incompetent recombinant retroviral particle
  • an infusion composition or other cell formulation for treating or preventing cancer or tumor growth comprising polynucleotides such as polynucleotide vectors, in illustrative embodiments RIPs, or modified cells, such as a modified lymphocytes, modified TILs, modified lymphocytes other than B cells, or modified T cells and/or modified NK cells.
  • polynucleotides such as polynucleotide vectors, in illustrative embodiments RIPs, or modified cells, such as a modified lymphocytes, modified TILs, modified lymphocytes other than B cells, or modified T cells and/or modified NK cells.
  • polynucleotides such as polynucleotide vectors, in illustrative embodiments RIPs, or modified cells, such as a modified lymphocytes, modified TILs, modified lymphocytes other than B cells, or modified T cells and/or modified NK cells of the delivery composition or infusion composition can include any of the aspects, embodiments, or subembodiments discussed above or elsewhere herein, for example that include nucleic acids that encode anti-idiotype polypeptides, as well as a CAR, an LE, a cytokine and/or a TCR.
  • a container such as a commercial container or package, or a kit comprising the same, comprising polynucleotides, such as polynucleotide vectors, for example RIPs, or modified cells, such as a modified lymphocytes, modified TILs, modified lymphocytes other than B cells, or modified T cells and/or modified NK cells according to any of the aspects and embodiments provided herein.
  • polynucleotides such as polynucleotide vectors, for example RIPs
  • modified cells such as a modified lymphocytes, modified TILs, modified lymphocytes other than B cells, or modified T cells and/or modified NK cells according to any of the aspects and embodiments provided herein.
  • the polynucleotides such as polynucleotide vectors, for example RIPs, or modified cells, such as a modified lymphocytes, modified TILs, modified lymphocytes other than B cells, or modified T cells and/or modified NK cells can comprise in their genome a polynucleotide comprising one or more nucleic acid sequences operatively linked to a promoter active in T cells and/or NK cells.
  • a nucleic acid sequence of the one or more nucleic acid sequences can encode an anti-idiotype polypeptide, an inhibitory RNA, a cytokine, a lymphoproliferative element and/or a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activating domain.
  • a nucleic acid sequence of the one or more nucleic acid sequences can encode one, two or more inhibitory RNA molecules directed against one or more RNA targets.
  • the container that contains the polynucleotides can be a cryopreservation infusion bag, tube, vial, well of a plate, or other vessel for storage of polynucleotides, such as polynucleotide vectors, for example RIPs, or modified cells, such as a modified lymphocytes, modified TILs, modified lymphocytes other than B cells, or modified T cells and/or modified NK cells.
  • polynucleotides such as polynucleotide vectors, for example RIPs
  • modified cells such as a modified lymphocytes, modified TILs, modified lymphocytes other than B cells, or modified T cells and/or modified NK cells.
  • some aspects provided herein comprise a container comprising polynucleotides, such as polynucleotide vectors, for example RIPs, or modified cells, such as a modified lymphocytes, modified TILs, modified lymphocytes other than B cells, or modified T cells and/or modified NK cells, wherein such biological include any nucleic acid(s) or other component(s) disclosed herein.
  • polynucleotides such as polynucleotide vectors, for example RIPs
  • modified cells such as a modified lymphocytes, modified TILs, modified lymphocytes other than B cells, or modified T cells and/or modified NK cells, wherein such biological include any nucleic acid(s) or other component(s) disclosed herein.
  • Such container in illustrative embodiments includes substantially pure polynucleotides, such as polynucleotide vectors, for example RIPs, or modified cells, such as a modified lymphocytes, modified TILs, modified lymphocytes other than B cells, or modified T cells and/or modified NK cells , sometimes referred to herein for shorthand, as substantially pure biologic.
  • substantially pure polynucleotides such as polynucleotide vectors, for example RIPs
  • modified cells such as a modified lymphocytes, modified TILs, modified lymphocytes other than B cells, or modified T cells and/or modified NK cells , sometimes referred to herein for shorthand, as substantially pure biologic.
  • a preparation and/or container of substantially pure biologic is sterile, and negative for mycoplasma, replication competent retroviruses, and adventitious viruses according to standard protocols (see e.g., “Viral Vector Characterization: A Look at Analytical Tools”; October 10, 2018 (available at https://cellculturedish.com/viral-vector-characterization-analytical-tools/)).
  • Exemplary methods for generating substantially pure biologies and cells for cell therapy are known. For example, for such methods with respect to RIPs, viral supernatants can be purified by a combination of depth filtration, TFF, benzonase treatment, diafiltration, and formulation.
  • a substantially pure biologic meets all of the following characteristics based on quality control testing results: a) negative for mycoplasma; b) endotoxin at less than 25 EU/ml, and in certain further illustrative embodiments, less than 10 EU/ml; c) absence of replication competent retroviruses detected of the same type as purposefully in the container (e.g., lentiviruses) detected; and d) absence of adventitious viruses detected;
  • the biologic is a viral particle, such as a retroviral particle
  • it meets the following quality control testing results: a) less than 1 pg host cell DNA/ viral TU, and in certain further illustrative embodiments, less than 0.3 pg / TU; b) less than 100 residual plasmid copies/ viral TU, and in certain further illustrative embodiments, less than 10 copies/viral TU of any plasmid used to make the recombinant retroviral particles; c) less than 1 ng HEK protein/ TU, and in certain further illustrative embodiments, less than 50 pg HEK protein /TU; and d) greater than 100 TU / ng P24 protein, and in certain further illustrative embodiments, greater than 10,000 TU /ng P24 protein.
  • Retroviral particles are typically tested against release specifications that include some or all of those provided above, before they are released to a customer. Potency of each particle may be defined on the basis of p24 viral capsid protein by ELISA, viral RNA genome copies by q-RT PCR, measurement of reverse transcriptase activity by qPCR-based product-enhanced RT (PERT) assay but can all be converted to infectious titer by measuring functional gene transfer Transducing Units (TUs) in a bioassay.
  • TUs functional gene transfer Transducing Units
  • Determination of infectious titer of purified bulk retrovirus material and finished product by bioassay and qPCR is an exemplary analytical test method for the determination of infectious titer of retroviruses.
  • An indicator cell bank (such as F1XT) may be grown for example in serum free media, seeded at 150,000 cells per well, followed by exposure to serial dilutions of the retrovirus product. Dilutions of purified retrovirus particles are made on indicator cells, for example from 1:200 to 1:1,600. A reference standard virus may be added for system suitability. Following 4 days of incubation with retrovirus, the cells are harvested, DNA extracted and purified.
  • a standard curve, for example from 100- 10,000,000 copies/ well, of human genome and unique retroviral genome sequence plasmid pDNA amplicons are used followed by addition of genomic DNA of the cell samples exposed to retrovirus particles.
  • the Cq values of both the retrovirus amplicon and the endogenous control such as humanRNAseP are extrapolated back to copies per reaction. From these values the integrated genome copy number is calculated.
  • indicator cells such as 293T have been characterized as being triploid, hence 3 copies of a single copy gene per cell should be utilized in the calculation.
  • TU Transducing Unit
  • Potency testing can include potency testing against release specifications with purity and specific activity.
  • potency release testing of final product can include measurement of the number of Transducing Units (TU) can be compared to viral particle quantity (e.g., by performing an ELISA against a viral protein, for example for lentivirus by performing a p24 capsid protein ELISA with a cutoff of at least 100, 1,000, 2,000 or 2,500 TU/ng p24), and CAR functionality, for example by measuring interferon gamma release by a reporter cell line exposed to gene modified cells.
  • TU Transducing Units
  • kits or isolated replication incompetent recombinant retroviral particle aspects herein that include a container of such retroviral particles, sufficient recombinant retroviral particles are present in the container to achieve an MOI (the number of Transducing Units, or TUs applied per cell) in a reaction mixture made using the retroviral particles, of between 0.1 and 50, 0.5 and 50, 0.5 and 20, 0.5 and 10, 1 and 25, 1 and 15, 1 and 10, 1 and 5, 2 and 15, 2 and 10, 2 and 7, 2 and 3, 3 and 10, 3 and 15, or 5 and 15 or at least 0.1, 0.5, 1, 2, 2.5, 3, 5, 10 or 15, or to achieve an MOI of at least 0.1, 0.5, 1, 2, 2.5, 3, 5, 10 or 15.
  • MOI the number of Transducing Units, or TUs applied per cell
  • the Transducing Units of virus particles provided in the kit should enable the use an MOI that prevents producing too many integrants in an individual cell, on average less than 3 lentigenome copies per cellular genome and more preferably 1 copy per cell.
  • MOI can be based on 1, 2.5, 5, 10, 20, 25, 50, 100, 250, 500, or 1,000 ml of reaction mixture assuming IxlO 6 target cells/ml, for example in the case of whole blood, assuming 1 x 10 6 PBMCs/ml of blood.
  • a container of retroviral particles can include between 1 x 10 5 and 1 x 10 9 , 1 x 10 5 and 1 x 10 8, 1 x 10 5 and 5 x 10 7 , 1 x 10 5 and 1 x 10 7 , 1 x 10 5 and 1 x 10 6 ; 5 x 10 5 and 1 x 10 9 ; 5 x 10 5 and 1 x 10 8 , 5 x 10 5 and 5 x 10 7 , 5 x 10 5 and 1 x 10 7 , 5 x 10 5 and 1 x 10 7 , 5 x 10 5 and 1 x 10 6 , or 1 x 10 7 and 1 x 10 9 , 1 x 10 7 and 5 x 10 7 , 1 x 10 6 and 1 x 10 7 , and 1 x 10 6 and 5 x 10 6 TUs.
  • the container can contain between 1 x 10 7 and 1 x 10 9 , 5 x 10 6 and 1 x 10 8 , 1 x 10 6 and 5 x 10 7 , 1 x 10 6 and 5 x 10 6 or between 5 xlO 7 and 1 xlO 8 retroviral Transducing Units.
  • such numbers of particles would support between 1 and 100 ml of blood at an MOI of between 1 and 10.
  • as little as 10 ml, 5 ml, 3 ml, or even 2.5 ml of blood can be processed for T cell and/or NK cell modification and optionally subcutaneous and/or intramuscular administration methods provided herein.
  • an advantage of the present methods is that in some illustrative embodiments, they require far fewer retroviral particle Transducing Units than prior methods that involve nucleic acids encoding a CAR, such as CAR-T methods.
  • Each container that contains retroviral particles can have, for example, a volume of between 0.05 ml and 5 ml, 0.05 ml and 1 ml, 0.05 ml and 0.5 ml, 0.1 ml and 5 ml, 0.1 ml and 1 ml, 0.1 ml and 0.5 ml, 0.1 and 10 ml, 0.5 and 10 ml, 0.5 ml and 5 ml, 0.5 ml and 1 ml, 1.0 ml and 10.0 ml, 1.0 ml and 5.0 ml, 10 ml and 100 ml, 1 ml and 20 ml, 1 ml and 10 ml, 1 ml and 5 ml, 1 ml and 2 ml, 2 ml and 20 ml, 2 ml and 10 ml, 2 ml and 5 ml, 0.25 ml to 10 ml, 0.25 to 5 ml, or 0.
  • the container such as the cryopreservation infusion bag
  • the container can hold 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, or 500 ml or less of blood.
  • the container, for example cry opreservation infusion bag can hold at least 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, or 500 ml of blood.
  • the container for example cryopreservation infusion bag can hold between 1, 2, 3, 4, 5, 10, 15, 20, 25, and 50 ml of blood on the low end of the range and 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, and 500 ml of blood on the high end of the range. In some embodiments, the container, for example cryopreservation infusion bagcan hold between 1, 2, 3, 4, 5, 10, 15, 20, 25, and 50 ml of blood on the low end of the range and 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, and 500 ml of blood on the high end of the range.
  • the container for example cry opreservation infusion bag can hold between 1 and 10 ml, 5 and 25 ml, 10 and 50 ml, 25 and 100 ml, 50 and 200 ml, or 100 and 500 ml of blood.
  • the container for example cryopreservation infusion bag can include heparin.
  • the container, for example cryopreservation infusion bag do not include heparin.
  • the number of cells delivered can be sufficient to provide between 1 x 10 5 cells and 1 x 10 9 , between 1 x 10 6 cells and 1 x 10 9 , or between 1 x 10 6 cells and 5 x 10 8 , for example CAR-positive viable T cells and/or NK cells per kg of body weight of the subject to which the cells are to be delivered.
  • the commercial container can include the aforementioned ranges x 50-150 kg, or 50- 100kg.
  • the commercial container includes between 1 x 10 7 and 1 x 10 11 cells, between 1 x 10 8 and 1 x 10 11 cells, or between 1 x 10 8 and 5 x 10 10 cells, for example CAR-positive viable T cells and/or NK cells, or in an illustrative embodiment, cells that are positive for an anti-idiotype extracellular recognition domain.
  • the polynucleotides encoding the anti-idiotype polypeptide and in illustrative embodiments the CAR are located in the genome of retroviral particles, typically substantially pure retroviral particles, according to any of the replication incompetent recombinant retroviral particle aspects and embodiments provided herein.
  • the replication incompetent recombinant retroviral particles in the kit comprise a polynucleotide comprising one or more transcriptional units operatively linked to a promoter active in T cells and/or NK cells, wherein the one or more first transcriptional units encode a first polypeptide comprising an anti-idiotype polynucleotide and a CAR or an LE and optionally encode a second polypeptide comprising the other of a CAR or an LE, according to any of the embodiments provided herein.
  • a kit provided herein can include a container containing the polynucleotides, such as polynucleotide vectors, for example RIPs, or modified cells, such as, modified lymphocytes, modified TILs, modified lymphocytes other than B cells, for example modified T cells and/or NK cells, and an accessory kit.
  • polynucleotides such as polynucleotide vectors, for example RIPs
  • modified cells such as, modified lymphocytes, modified TILs, modified lymphocytes other than B cells, for example modified T cells and/or NK cells, and an accessory kit.
  • the accessory kit components can include one or more of the following: a) one or more containers containing a delivery solution compatible with, in illustrative embodiments effective for, and in further illustrative embodiments adapted for subcutaneous and/or intramuscular administration as provided herein; b) one or more containers of hyaluronidase as provided herein; c) one or more blood bags such as a blood collection bag, in illustrative embodiments comprising an anticoagulant in the bag, or in a separate container, a blood processing buffer bag, a blood processing waste collection bag, and a blood processing cell sample collection bag; d) one or more sterile syringes compatible with, in illustrative embodiments effective for, and in further illustrative embodiments adapted for, subcutaneous or intramuscular delivery of T cells and/or NK cells; e) a T cell activation element as disclosed in detail herein, for example anti-CD3 provided in solution in the container containing the retroviral particle, or in
  • recombinant retroviral particles comprising one or more second transcriptional units operatively linked to a promoter active in T cells and/or NK cells, wherein the one or more second transcriptional units encode a polypeptide comprising a second CAR directed against a different target epitope, and in certain embodiments a different antigen, in illustrative embodiments found on a same target cancer cell (e.g.
  • B cell B cell
  • k one or more containers containing a cognate antigen for the first CAR and/or the second CAR encoded by the nucleic acids (e.g., retroviral particles); and l) instructions, either physically or digitally associated with other kit components, for the use thereof, for example for modifying T cells and/or NK cells, for delivering modified T cells and/or NK cells to a subject subcutaneously or intramuscularly, and/or for treating tumor growth or cancer in a subject.
  • the nucleic acids e.g., retroviral particles
  • kits that include an antigen or a cognate antigen, less than 50%, 40%, 30%, 20%, 10%, 5%, or 1% of the polypeptides in the kit are non-human, i.e., produced from non-human sources.
  • the kit may be a single-pack/use kit, but in other embodiments the kit is a multi-pack or multi-use kit for the processing of more than one blood sample from contacting with nucleic acids encoding a CAR optionally thru subcutaneous administration.
  • a container of nucleic acids encoding a CAR (and optionally a paired container of nucleic acids encoding a second CAR in certain embodiments) in the kit is used for one performance of a method for modifying T cells and/or NK cells and optionally subcutaneous administration.
  • the container(s) containing nucleic acids encoding a CAR and optionally a second CAR is typically stored and shipped frozen.
  • a kit can include sufficient containers (e.g., vials) of nucleic acids encoding a CAR (and optionally paired containers encoding a second CAR in certain embodiments) for 1, 2, 3, 4, 5, 6, 10, 12, 20, 24, 50 and 100 performances of a method for modifying a T cell and/or NK cell provided herein, and thus can include 1 , 2, 3, 4, 5, 6, 10, 12, 20, 24, 50 and 100 containers (e.g., vials) of nucleic acids encoding the CAR (e.g., retroviral particles), and similarly is considered a 1, 2, 3, 4, 5, 6, 10, 12, 20, 24, 50 and 100 pack, performance, administration or X kit, respectively.
  • accessory components in the kit would be provided for similar numbers of performances of a method for modifying T cells and/or NK cells and optionally subcutaneous administration, using the kit.
  • the one or more leukoreduction filtration assemblies typically include(s) one or a plurality of leukoreduction filters or leukoreduction filter sets, each typically within a filter enclosure, as well as a plurality of connected sterile tubes connected or adapted to be connected thereto, and a plurality of valves connected or adapted to be connected thereto, that are adapted for use in a singleuse closed blood processing system.
  • leukoreduction filtration assembly for each container of nucleic acid encoding a CAR in a kit.
  • a 20-pack kit in illustrative embodiments, includes 20 vials of nucleic acids encoding a CAR and 20 leukoreduction filtration assemblies.
  • a kit herein comprises one or a plurality of containers containing nucleic acids and one or more leukoreduction filtration assemblies.
  • Such a kit can optionally be intended to be used for administration to a subject via any route including for example, infusion or in illustrative embodiments intramuscular and/or in further illustrative embodiments, subcutaneous delivery.
  • such a kit optionally includes other accessory components that are intended to be used with such route of administration.
  • the one or more containers of subcutaneous or intramuscular delivery solution is typically sterile and can include a total combined volume, or individually per container, of 100 ml to 5 L, 1 ml to 1 L, 1 ml to 500 ml, 1 ml to 250 ml, 1 ml to 200 ml, 1 ml to 100 ml, 1 ml to 10 ml, or 1 ml to 5 ml; 5 ml to 1 L, 5 ml to 500 ml, 5 ml to 250 ml, 5 ml to 100 ml, 5 ml to 10 ml, or approximately 5 ml.
  • the kit comprises a plurality of containers of subcutaneous delivery solution, with each container having a volume of between 10 ml and 200 ml, 10 ml and 100 ml, 1 ml and 20 ml, 1 ml and 10 ml, 1 ml and 5 ml, 1 ml and 2 ml, 2 ml and 20 ml, 2 ml and 10 ml, 2 ml and 5 ml, 0.25 ml to 10 ml, 0.25 to 5 ml, or 0.25 to 2 ml.
  • a 20-pack kit in illustrative embodiments, includes 20 vials of nucleic acids encoding a CAR and 20 containers of sterile delivery solution.
  • accessory components of the kit can further include one or more of the following: a. one or more containers containing a delivery solution adapted for, compatible with, and/or effective for, intravenous or intraperitoneal administration as provided herein; and b. Instructions, either physically or digitally associated with other kit components, for the use thereof, for example for delivering modified T cells and/or NK cells to a subject intravenously or intraperitoneally.
  • kits for modifying a T cell or NK cell wherein the use of the kit includes: contacting the T cell or NK cell ex vivo with the replication incompetent recombinant retroviral particle, wherein the replication incompetent recombinant retroviral particle includes a pseudotyping element on a surface and a T cell activation element on the surface, wherein said contacting facilitates transduction of the T cell or NK cell by the replication incompetent recombinant retroviral particle, thereby producing a modified and in illustrative embodiments genetically modified T cell or NK cell.
  • the T cell or NK cell can be from a subject.
  • the T cell activation element can be membrane-bound.
  • the contacting can be performed for between 1, 2, 3, 4, 5, 6, 7, or 8 hours on the low end of the range and 4, 5, 6, 7, 8, 10, 12, 15, 18, 21, and 24 hours on the high end of the range, for example, between 1 and 12 hours.
  • the replication incompetent recombinant retroviral particle for use in the manufacture of a kit can include any of the aspects, embodiments, or subembodiments discussed elsewhere herein.
  • kits such as a commercial container or package, or a kit comprising the same, comprising isolated packaging cells, in illustrative embodiments isolated packaging cells from a packaging cell line, according to any of the packaging cell and/or packaging cell line aspects provided herein.
  • the kit includes additional containers that include additional reagents such as buffers or reagents used in methods provided herein.
  • additional reagents such as buffers or reagents used in methods provided herein.
  • any replication incompetent recombinant retroviral particle provided herein in any aspect, in the manufacture of a kit for modifying and in illustrative embodiments genetically modifying a T cell or NK cell according to any aspect provided herein.
  • any packaging cell or packaging cell line provided herein in any aspect, in the manufacture of a kit for producing the replication incompetent recombinant retroviral particles according to any aspect provided herein.
  • polynucleotides referred to herein as “self-driving CARs” that encode a membrane-bound lymphoproliferative element whose expression in a T cell or NK cell is under the control of an inducible promoter that is induced by the binding of an antigen to an extracellular binding pair member polypeptide that is expressed by the T cell or NK cell and is functionally linked to a intracellular activating domain, for example a CD3 zeta intracellular activating domain or any of the intracellular activating domains disclosed elsewhere herein.
  • an antiidiotype polypeptide is co-expressed by the T cell or NK cell to provide additional functional optionality for self-driving CARs.
  • the co-expressed anti-idiotype polypeptide is a safety switch.
  • such a binding pair member polypeptide is a CAR.
  • such a binding pair member polypeptide is a TCR.
  • polynucleotides that include an inducible promoter operably linked to a nucleic acid encoding a membrane-bound lymphoproliferative element, that is induced by CAR-binding to its target.
  • Self-driving CAR-T cells genetically modified or transduced T cells referred to herein as “self-driving CAR-T cells” that include a self-driving CAR.
  • Any of the embodiments that include a self-driving CAR-T cell could include a “self-driving CAR NK cell,” which is a genetically modified or transduced NK cell that includes a self-driving CAR.
  • the self-driving CAR NK cell is present in addition to the self-driving CAR-T cell.
  • the self-driving CAR NK cell is present instead of the self-driving CAR-T cell.
  • Various embodiments that include a self-driving CAR are disclosed in the Exemplary Embodiments section herein and can be combined with any of the embodiments or details of this section.
  • Methods provided herein in illustrative aspects include methods for modifying T cells and/or NK cells, or related methods of making cell formulations, that include contacting blood cells comprising lymphocytes (e.g., NK cells and/or T cells) ex vivo in a reaction mixture, with recombinant vectors such as replication incompetent recombinant retroviral particles, that are or include polynucleotides that encode a CAR.
  • the reaction mixture includes a T cell activation element, either in solution or on the surface of the recombinant retroviral particles, to facilitate genetic modification of T cells in the reaction mixture
  • Some of the methods provided herein include an optional step where blood is collected (110) from a subject.
  • Blood can be collected or obtained from a subject by any suitable method known in the art as discussed in more detail herein.
  • the blood can be collected by venipuncture, apheresis or any other blood collection method by which a sample of blood is collected.
  • the volume of blood collected is between 1 and 120 ml.
  • the volume of blood collected is between 1 ml and 25 ml.
  • lymphocytes e.g., T cells and/or NK cells
  • the lymphocytes are contacted with encapsulated nucleic acid vectors (e.g. replication incompetent retroviral particles) in a reaction mixture.
  • this contacting, and the reaction mixture in which the contacting occurs takes place within a closed cell processing system, as discussed in more detail herein.
  • a closed processing system and method used in some aspects and embodiments of systems and methods provided herein can be any system and method known in the art.
  • the system or method can be a traditional closed cell processing system and method, or a system or method referred to herein as a “more recent” method or system (See e.g., WO2018/136566 and WO2019/055946, each incorporated herein by reference in their entirety).
  • traditional closed cell processing methods that involve genetic modification and/or transductions of lymphocytes ex vivo, especially in methods for autologous cell therapy, many steps occur over days, such as PBMC enrichment(s), washing(s), cell activation, transduction, expansion, collection, and optionally reintroduction.
  • PBMC enrichment(s) washing(s)
  • cell activation transduction
  • expansion expansion
  • collection and optionally reintroduction
  • the T cell and/or NK cell activation element is associated with surfaces of retroviral particles present in the reaction mixture.
  • a rapid point-of-care (rPOC) autologous cell therapy method is used in a rapid point-of-care (rPOC) autologous cell therapy method.
  • rPOC rapid point-of-care
  • PBMC enrichment step/procedure 120A
  • cell counting, transfer and media addition which takes at least around 45 additional minutes before lymphocytes are contacted with retroviral particles to form a transduction reaction mixture (130A).
  • lymphocytes are typically washed away from retroviral particles that remain in suspension (140A), for example using a Sepax, and collected by resuspending the PBMCs in a delivery solution (150A) to form a cell formulation typically in an infusion bag for reinfusion, a syringe for injection, or cryopreservation vial for storage (160A).
  • PBMC enrichment procedures typically involve ficoll density gradients and centrifugal (e.g., centrifugation) or centripetal (e.g., Sepax) forces or use leukophoresis to enrich PBMCs.
  • modified lymphocytes e.g., T cells and/or NK cells
  • a solution e.g., T cells and/or NK cells
  • reintroduced into a subject by subcutaneous administration, delivery, or injection.
  • retroviral particles such as those exemplified in FIG.
  • resulting cell formulations are optionally administered (e.g., readministered) into a subject.
  • FIG. ID where a PBMC enrichment procedure is not used after lymphocytes are contacted with retroviral particles, cell formulations produced there can be reintroduced back into a subject using subcutaneous or intramuscular administration.
  • cell formulations as well as delivery solutions (i.e., excipients) for making such cell formulations, that are compatible with, in illustrative embodiments effective for, and in further illustrative embodiments adapted for subcutaneous delivery.
  • additional blood cells especially neutrophils
  • cell processing filters to concentrate and/or wash lymphocytes, such as HemaTrate filters
  • a subcutaneous formulation of retrovirus reconstituted with total nucleated cells on a lymphoreduction filter may contain, in addition to lymphocytes, neutrophils (or more generally granulocytes).
  • the cell formulation comprises neutrophils, B cells, monocytes, red blood cells, basophils, eosinophils, and/or macrophages together with modified T cells (CAR-T cells) and/or NK cells (CAR-NK cells).
  • CAR-T cells modified T cells
  • CAR-NK cells CAR-NK cells
  • a subcutaneous or intramuscular formulation and administration are advantageous over intravenous formulation and administration because a formulation (suspension) of retrovirus reconstituted with lymphocytes may further comprise cellular aggregates and express adhesion receptors that may introduce pulmonary congestion with intravenous delivery.
  • subcutaneous administration Methods for subcutaneous administration are well known in the art and typically involve administration into the fat layer under the skin. It should be noted that it is contemplated that any embodiment herein that involves subcutaneous delivery, can instead be intramuscular delivery, which is delivery into the muscle, intradermal, or intratumoral delivery. In some embodiments, subcutaneous administrations can be performed in the upper thigh, upper arm, abdomen, or upper buttocks of a subject. Subcutaneous administration is distinguishable from intraperitoneal administration, which penetrates through the fatty layer used in subcutaneous administration and delivers a formulation or drug into the peritoneum of the subject.
  • a method of administering modified cells to a subject which can include before delivering the modified cells to the subject, a step of transducing, transfecting, genetically modifying, and/or modifying the cells.
  • the cells can be lymphocytes, such as a (typically a population of) peripheral blood mononuclear cells (PBMCs), typically T cells and/or an NK cells, and in certain illustrative embodiments resting T cells and/or resting NK cells.
  • PBMCs peripheral blood mononuclear cells
  • NK cells typically T cells and/or an NK cells
  • the transducing, transfecting, modifying and/or genetically modifying step can include contacting the lymphocytes with a population of recombinant nucleic acid/polynucleotide vectors, which in certain illustrative embodiments include nucleic acids encoding an anti-idiotype polypeptide, and which in illustrative embodiments are replication incompetent recombinant retroviral particle (RIPs), wherein said contacting (and incubation under contacting conditions) facilitates membrane association, membrane fusion or endocytosis, and optionally transduction or transfection of the cells, for example resting T cells and/or NK cells by the recombinant nucleic acid vectors, thereby producing the modified and in illustrative embodiments genetically modified T cells and/or NK cells.
  • nucleic acid/polynucleotide vectors which in certain illustrative embodiments include nucleic acids encoding an anti-idiotype polypeptide, and which in illustr
  • the RIP typically comprises a fusogenic element and a binding element, which can be part of a pseudotyping element, on its surface.
  • T cells and/or NK cells are activated before being contacted by the RIP or other polynucleotide vector.
  • pre-activation of the T cell and/or NK cell is not required, and an activation element, which can be any activation element provided herein, is present in a reaction mixture in which the contacting takes place.
  • the activation element is present on a surface of the replication incompetent recombinant retroviral particle.
  • the activation element is anti-CD3, such as anti-CD3 scFv, or anti-CD3 scFvFc.
  • Many of the method aspects provided herein include one or more of the following steps: 1) a step of collecting blood from a subject; 2) a step of contacting cells, such as NK cells and/or in illustrative embodiments T cells, which can be from the collected blood, with a recombinant vector (typically many copies thereof), in illustrative embodiments a RIP, encoding a CAR and/or a lymphoproliferative element, in a reaction mixture, where the contacting can include an optional incubation; 3) a step of washing unbound recombinant vector away from the cells in the reaction mixture; 4) a step of collecting modified cells, such as modified NK cells and/or in illustrative embodiments modified T cells in a solution, which in illustrative embodiments can be a delivery solution, to form a cell suspension, that in illustrative embodiments is a cell formulation; and 5) a step of delivering the cell formulation to a subject, in illustrative embodiments
  • the reaction mixture includes unfractionated whole blood or includes one or more cell type that is not a PBMC, and can include all or many cell types found in whole blood, including total nucleated cells (TNCs).
  • the recombinant vector comprises a self-driving CAR, which encodes both a CAR and a lymphoproliferative element.
  • the collected, unfractionated blood/isolated cells are passed through a leukoreduction filter to isolate TNCs on top of the filter; replication incompetent recombinant retroviral particles are added to the TNCs on top of the leukoreduction filter to a total reaction mixture volume of 500 pl to 10 ml to form a reaction mixture and initiate contacting; the reaction mixture is optionally incubated for any of the contacting times provided herein, as a non-limiting example, for 1-4 hours; the non-associated replication incompetent recombinant retroviral particles are washed away from cells in the reaction mixture by filtering the reaction mixture with 10 to 120 ml of wash solution; and the cells, including modified T cells and NK cells, which are retained on the TNC filter, are
  • any methods used in any aspects provided herein can include a step for modifying and in illustrative embodiments genetically modifying lymphocytes, PBMCs, and in illustrative embodiments NK cells and/or in further illustrative embodiments, T cells, can include a step of collecting blood from a subject.
  • the blood includes blood components including blood cells such as lymphocytes (e.g., T cells and NK cells) that can be used in methods and compositions provided herein.
  • the subject is a human subject afflicted with cancer (i.e., a human cancer subject). It is noteworthy that certain embodiments do not include such a step.
  • blood can be collected or obtained from a subject by any suitable method known in the art as discussed in more detail herein, and as such the collected blood or blood-derived component can be referred to as a “blood-derived product” and typically is a “peripheral blood-derived product,” since typically it is isolated from peripheral blood.
  • the blood-derived product can be collected by venipuncture or any other blood collection method known in the art, by which a sample of unfractionated whole blood is collected in a vessel, for example a blood bag, or by which leukocytes and lymphocytes are isolated from blood, such as by apheresis (e.g., leukapheresis or lymphoplasmapheresis).
  • the volume of blood (e.g., unfractionated whole blood) collected is between 1 and 5 ml, 5 and 10 ml, 10 and 15 ml, 15 and 20 ml, 20 and 25 ml, 5 and 25 ml, 25 ml and 250 ml, 25 ml and 125 ml, 50 ml and 100 ml, or 50 ml and 250 ml, 75 ml and 125 ml, 90 ml and 120 ml, or between 95 and 110 ml.
  • the volume of blood collected can be between 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 600, 700, 800, or 900 ml on the low end of the range and 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 600, 700, 800, or 900 ml or 1 L on the high end of the range.
  • the volume of blood collected is less than 250 ml, 100 ml, 75 ml, 20 ml, 15 ml, 10 ml, or 5 ml.
  • lymphocytes e.g., T cells and/or NK cells
  • T cells and/or NK cells can be obtained by apheresis.
  • the volume of blood taken and processed during apheresis is between 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1, 1.25, or 1.5 total blood volumes of a subject on the low end of the range and 0.6, 0.7, 0.75, 0.8, 0.9, 1, 1.25, 1.5 1.75, 2, 2.25, or 2.5 total blood volumes of a subject on the high end of the range, for example between 0.5 and 2.5, 0.5 and 2, 0.5 and 1.5, or between 1 and 2 total blood volumes.
  • the total blood volume of a human typically ranges from 4.5 to 6 L and thus much more blood is typically taken and processed during apheresis than if unfractionated whole blood is collected.
  • target blood cells e.g., T cells
  • target blood cells e.g., T cells
  • target blood cells therein would be processed according to a method provided herein, which in certain illustrative embodiments results in the target blood cells becoming modified, genetically modified, and/or transduced.
  • apheresis e.g., leukapheresis or lymphoplasmapheresis
  • a cell fraction comprising T cells and/or NK cells (e.g., to provide a leukopak or a lymphoplasmapak)
  • NK cells e.g., to provide a leukopak or a lymphoplasmapak
  • Such reaction mixture can be used in any method herein.
  • apheresis e.g., leukapheresis or lymphoplasmapheresis
  • blood cells e.g., White blood cells or lymphocytes
  • lymphocytes e.g. T cells and/or NK cells
  • a population of cells such as lymphocytes (e.g. T cells and/or NK cells) are typically contacted with many copies of a recombinant vector, which in some embodiments are copies of a non-viral vector, and in illustrative embodiments are identical RIPs, in a reaction mixture.
  • the contacting in any embodiment provided herein, can be performed for example in a chamber of a closed system adapted for processing of blood cells, for example within a blood bag, as discussed in more detail herein.
  • the blood bag can have 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, or 500 ml or less of blood during the contacting.
  • the blood bag can have at least 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, or 500 ml of blood during the contacting.
  • the blood bag can have between 1, 2, 3, 4, 5, 10, 15, 20, 25, and 50 ml of blood on the low end of the range and 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, and 500 ml of blood on the high end of the range during the contacting.
  • the blood bag can have between 1 and 10 ml, 5 and 25 ml, 10 and 50 ml, 25 and 100 ml, 50 and 200 ml, or 100 and 500 ml of blood during the contacting.
  • the mixture inside the blood bag can include an anti-coagulant such as heparin.
  • the mixture inside the blood bag does not include an ant-coagulant, or does not include heparin.
  • the transduction reaction mixture can include one or more buffers, ions, and a culture media.
  • lentiviral particles in certain exemplary reaction mixtures provided herein, between 0.1 and 50, 0.5 and 50, 0.5 and 20, 0.5 and 10, 1 and 25, 1 and 15, 1 and 10, 1 and 5, 2 and 15, 2 and 10, 2 and 7, 2 and 3, 3 and 10, 3 and 15, or 5 and 15, multiplicity of infection (MOI); or at least 1 and less than 6, 11, or 51 MOI; or in some embodiments, between 5 and 10 MOI units of replication incompetent recombinant retroviral particles are present.
  • the MOI can be at least 0.1, 0.5, 1, 2, 2.5, 3, 5, 10 or 15.
  • compositions and methods for transducing lymphocytes in blood in certain embodiments higher MOI can be used than in methods wherein PBMCs are isolated and used in the reaction mixtures.
  • illustrative embodiments of compositions and methods for transducing lymphocytes in whole blood assuming IxlO 6 PBMCs/ml of blood, can use retroviral particles with an MOI of between 1 and 50, 2 and 25, 2.5 and 20, 2.5 and 10, 4 and 6, or about 5, and in some embodiments between 5 and 20, 5 and 15, 10 and 20, or 10 and 15.
  • the reaction mixture of the contacting step included in some methods provided herein, or reaction mixture aspects in some embodiments comprises at least 10% unfractionated whole blood (e.g. at least 10%, 20%, 25%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% whole blood) and optionally an effective amount of an anticoagulant; or the reaction mixture further comprises at least one additional blood or blood preparation component that is not a PBMC, for example the reaction mixture comprises an effective amount of an anticoagulant and one or more blood preparation component that is not a PBMC.
  • a percentage of whole blood is the percent by volume of a reaction mixture that was made using unfractionated whole blood.
  • the percentage of whole blood in the reaction mixture is the volume of whole blood by the total volume of the reaction mixture times 100.
  • such blood or blood preparation component that is not a PBMC is one or more (e.g., at least one, two, three, four, or five) or all of the following additional components: a) erythrocytes, wherein the erythrocytes comprise between 1 and 60% of the volume of the reaction mixture; b) neutrophils, wherein the neutrophils comprise at least 10% of the white blood cells in the reaction mixture, or wherein the reaction mixture comprises at least 10% as many neutrophils as T cells; c) basophils, wherein the basophils comprise at least 0.05% of the white blood cells in the reaction mixture; d) eosinophils, wherein the reaction mixture comprises at least 0.1% of the white blood cells in the reaction mixture; e) plasma, wherein the plasma comprises at least 1% of the volume of the reaction mixture; and f) an anticoagulant,
  • the percentage is based on volume.
  • at least 25% of the volume of a reaction mixture can be whole blood.
  • at least 25 ml of 100 ml of such reaction mixture would be whole blood.
  • TCR complex including TCRot, TCR
  • the extent of this dimming increases as the concentration of a given gene vector is increased in the reaction mixture and correlates with the ability of the gene vector to activate and enter cells.
  • internalization of other surface polypeptides after binding to polypeptides on the surface of a gene vector results in dimming of the surface polypeptide on the cell being contacted with the gene vector and may be common during transduction using other binding polypeptides.
  • a percent reduction in surface polypeptide expression on cells contacted with a gene vector comprising a binding polypeptide compared to surface polypeptide expression on cells not contacted with the gene vector comprising a binding polypeptide is used to quantitate the potency of a gene vector and determine the appropriate dose of gene vector used to modify a population of cells.
  • a percent reduction in surface TCR complex expression on cells contacted with a gene vector compared to surface TCR complex expression on cells not contacted with the gene vector is used to quantitate the potency of a gene vector and determine the appropriate dose of gene vector used to modify a population of cells.
  • a “Dimming Unit” is the amount of gene vector (e.g.
  • RIR retroviral particles that reduces the surface expression of a surface polypeptide in 1 ml of a cell mixture after contacting with the gene vector for 4 hours at 37 °C and 5% CO2 by 50% compared to the surface expression of the surface polypeptide in the cell mixture under similar conditions but not contacted with the gene vector.
  • the surface polypeptide is typically a binding partner of a binding polypeptide present on the surface of the gene vector.
  • the surface polypeptide is a TCR complex polypeptide.
  • the TCR complex polypeptide is CD3D, CD3E, CD3G, CD3Z, TCRa, or TCR[3.
  • the binding partner is CD3 and the binding polypeptide is anti-CD3.
  • the ability of a polynucleotide vector to reduce surface expression of a surface polypeptide should be determined for each preparation of a polynucleotide vector. In some embodiments, the ability of a polynucleotide vector to reduce surface expression of a surface polypeptide is determined based on target cell number. In some embodiments, the ability of a polynucleotide vector to reduce surface expression of a surface polypeptide is based on the volume the cells.
  • the reduction of surface expression of a surface polypeptide can be referred to as dimming the surface polypeptide.
  • CD3 is dimmed on that cell and the cell can be called CD3-, even though the cell may still contain CD3 not expressed on its surface.
  • T cells that temporarily internalize and dim CD3 are T cells and will eventually re-express CD3 on their cell surfaces such that they are again CD3+.
  • a method for determining an amount of a polynucleotide vector preparation to dim surface expression of a surface polypeptide by a dimming percentage on cells in a dimming volume comprising: a) forming a plurality of reaction mixtures comprising a plurality of volumes of the polynucleotide vector preparation and a plurality of volumes of a cell mixture, wherein at least two of the reaction mixtures in the plurality of reaction mixtures comprise different volumes of the polynucleotide vector preparation and/or the cell mixture, wherein the cell mixture comprises a plurality of cells comprising the surface polypeptide on their surfaces, and wherein the polynucleotide vector preparation comprises a plurality of polynucleotide vectors comprising a binding polypeptide on their surfaces capable of binding the surface polypeptide; b) incubating the reaction mixtures; c) measuring the surface expression of the surface polypeptide in the reaction mixtures and in an uncontacted
  • the amounts of the cell mixture in the reaction mixtures is based on volume. In some embodiments, amounts of the cell mixture in the reaction mixtures is based on numbers of target cells.
  • the polynucleotide vector preparation is a viral preparation. In illustrative embodiments, the viral preparation is a replication incompetent recombinant retroviral particle preparation.
  • the dimming percentage (percentage of cells dimmed) is 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, or 97%. In illustrative embodiments, the dimming percentage is at least or about 80%, 85%, 90%, or 95%.
  • the dimming volume is 0.25 ml, 0.5 ml, 0.75 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 10 ml, 15 ml, 20 ml, or 25 ml.
  • the surface polypeptide can be CD3D, CD3E, CD3G, CD3Z, TCRa, TCR , CD16A, NKp46, 2B4, CD2, DNAM, or NKG2C, NKG2D, NKG2E, NKG2F, and/or NKG2H.
  • the surface polypeptide is a TCR complex polypeptide.
  • the TCR complex polypeptide is CD3D, CD3E, CD3G, CD3Z, TCRa, or TCR[3.
  • the surface polypeptide is CD3E.
  • the binding polypeptide can be any of the activation elements disclosed in the Activation Elements section herein. In such embodiments, the surface polypeptide can be the binding partner of the activation element.
  • the cell mixture is whole blood.
  • the cell mixture has been subjected to a red blood cell depletion procedure.
  • the whole blood is collected from a healthy subject, e.g., a subject that does not have or is not known or suspected to have a disease, disorder, or condition associated with associated with an elevated expression of an antigen.
  • the whole blood is collected from a subject with a disease, disorder, or condition associated with associated with an elevated expression of an antigen, wherein the polynucleotide vector will be administered to the subject or other subjects with the disease disorder, or condition.
  • the whole blood is collected from each subject and the Dimming Units are calculated for each subject individually.
  • the reaction mixtures can be incubated for less than or about 24, 12, 10, 8, 6, 4, or 2 hours or 60, 45, 30, 15, 10, or 5 minutes, or for just an initial contacting. In some embodiments, the reaction mixtures can be incubated for between 10 minutes and 24 hours, or between 10 minutes and 8 hours, or for between 1 hour and 8 hours, or for between 1 hour and 6 hours, or in illustrative embodiments, for between 3.5 and 4.5 hours or for 4 hours. In some embodiments, the reaction mixtures can be incubated at about 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 37 °C, or 42 °C. In some embodiments, the reaction mixtures are incubated without CO2. In illustrative embodiments, the reaction mixtures are incubated with 5% CO2.
  • the surface expression of the surface polypeptide is measured by fluorescence-activated cell sorting (FACS) method.
  • FACS fluorescence-activated cell sorting
  • the antibody used in a FACS method is GMP.
  • a CD3 antibody is used to determine surface expression of the surface polypeptide.
  • the CD3 antibody is UCHT1, OKT-3, HIT3A, TRX4, X35-3, VIT3, BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, Fl 11409, CEB-T3.4.2, TR-66, TR66.opt, HuM291, WT31, WT32, SPv-T3b, 11D8, XIII-141, XIII46, XIII-87, 12F6, T3/RW2-8C8, T3/RW24B6, OKT3D, M-T301, SMC2, F101.01, and SK7.
  • the CD3 antibody is PerCP Mouse Anti-Human CD3 - Clone SK7 (BD, 347344).
  • cells present in the cell mixture are separated from unbound polynucleotide vector in the incubated reaction mixture.
  • the polynucleotide vector preparation is a replication incompetent recombinant retroviral particle preparation
  • the dimming percentage is 50%
  • the dimming volume is 1 ml
  • the surface polypeptide is CD3
  • the cell mixture is whole blood collected from a healthy subject
  • the reaction mixture is incubated for 4 hours at 37 °C and 5% CO2 and the method is used to calculate Dimming Units.
  • Such methods can be used to determine the amount of retroviral particles in a polynucleotide vector preparation that reduces surface polypeptide expression on cells by a specific percentage. This amount can then be used to determine an amount of the preparation of retroviral particles to use for subsequent transductions of whole blood, isolated PBMCs, or isolated TNCs.
  • the amount of a preparation of polynucleotide vector, for example replication incompetent recombinant retroviral particles, to add to the lymphocytes can be determined using the method above.
  • Dimming Units can be used in any of the aspects or embodiments herein that include a contacting step to determine the amount of polynucleotide vector to add. As 1 DU of polynucleotide vector reduces the surface expression of the surface polypeptide by 50% in a 1 ml volume of cells, 10 DUs of polynucleotide vector reduces the surface expression of the surface polypeptide by 50% in 10 ml of a cell mixture.
  • sufficient DUs are added to a volume of cells to reduce surface expression of the surface polypeptide, for example CD3, by greater than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, or 97% after contacting with the polynucleotide vector compared to the surface expression of the surface polypeptide in the cell mixture under similar conditions but not contacted with the polynucleotide vector.
  • sufficient DUs are added to a volume of cells to reduce surface expression of the surface polypeptide by greater than 80%, 85%, 90%, or 95% after contacting with the polynucleotide vector compared to the surface expression of the surface polypeptide in the cell mixture under similar conditions but not contacted with the polynucleotide vector.
  • at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19 or 20 DU are added per ml of cell mixture.
  • between 5 and 20 DU, 5 and 15 DU, 10 and 20 DU, or 13 and 18 DU are added per ml of cell mixture.
  • the target cells are lymphocytes, for example T cells or NK cells.
  • the cells are in whole blood, isolated PBMCs, or isolated TNCs.
  • the cells are the remaining fraction of whole blood after lysing red blood cells.
  • sufficient DUs are added to dim a population of cells a specific percentage, for example, to dim CD3 on a population of T cells by greater than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, or 97%.
  • sufficient dimming units of a polynucleotide vector, and in illustrative embodiments RIP are present to increase the percentage of surface dimmed surface polypeptide, and in illustrative embodiments dimmed surface CD3-, in a population of cells, and in illustrative embodiments T cells, to at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, or 97%.
  • the composition including cells can include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19 or 20 DU per ml of the cells, for example at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19 or 20 DU per ml of blood, cell formulation, population if cells.
  • this contacting, and the reaction mixture in which the contacting occurs takes place within a closed cell processing system, as discussed in more detail herein.
  • a packaging cell and in illustrative embodiments a packaging cell line, and in particularly illustrative embodiments a packaging cell provided in certain aspects herein, can be used to produce the replication incompetent recombinant retroviral particles.
  • the cells in the reaction mixture can be PBMCs or TNCs, and/or in reaction mixture aspects herein that provide compositions and methods for transducing lymphocytes in whole blood, an anticoagulant and/or an additional blood component, including additional types of blood cells that are not PBMCs, can be present as discussed herein.
  • the reaction mixture can essentially be whole blood, and typically an anticoagulant, retroviral particles, and a relatively small amount of the solution in which the retroviral particles were delivered to the whole blood.
  • lymphocytes including NK cells and T cells, can be present at a lower percent of blood cells, and at a lower percentage of white blood cells, in the reaction mixture than methods that involve a PBMC enrichment procedure before forming the reaction mixture.
  • T cells can be for example, between 10, 20, 30, or 40% of the lymphocytes of the reaction mixture on the low end of the range, and between 40, 50, 60, 70, 80, or 90% of the lymphocytes of the reaction mixture on the high end of the range.
  • T cells comprise between 10 and 90%, between 20 and 90%, between 30 and 90%, between 40 and 90%, between 40 and 80%, or between 45% to 75% of the lymphocytes.
  • NK cells can be present at between 1, 2, 3, 4, or 5% of the lymphocytes of the reaction mixture on the low end of the range, and between 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14% of the lymphocytes of the reaction mixture on the high end of the range.
  • T cells comprise between 1 and 14%, between 2 and 14%, between 3 and 14%, between 4 and 14%, between 5 and 14%, between 5 to 13%, between 5 to 12%, between 5 to 11% or between 5 to 10% of the lymphocytes of the reaction mixture.
  • T cells can be at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the reaction mixture.
  • composition and method aspects for transducing lymphocytes in whole blood typically do not involve any blood fractionation such as a PBMC enrichment step of a blood sample, before lymphocytes from the blood sample are contacted with recombinant nucleic acid vectors, for example retroviral particles, in the reaction mixtures disclosed herein for those aspects.
  • lymphocytes in unfractionated whole blood are contacted with recombinant retroviral particles.
  • neutrophils/granulocytes are separated away from other blood cells before the cells are contacted with replication incompetent recombinant retroviral particles.
  • peripheral blood mononuclear cells PBMCs
  • PBLs peripheral blood lymphocytes
  • T cell and/or NK cells are isolated away from other components of a blood sample using for example, a PBMC enrichment procedure, before they are combined into a reaction mixture with retroviral particles.
  • a PBMC enrichment procedure is a procedure in which PBMCs are enriched at least 25-fold, and typically at least 50-fold from other blood cell types. For example, it is believed that PBMCs make up less than 1% of blood cells in whole blood. After a PBMC enrichment procedure, at least 30%, and in some examples as many as 70% of cells isolated in the PBMC fraction are PBMCs. It is possible that even higher enrichment of PBMCs is achieved using some PBMC enrichment procedures. Various different PBMC enrichment procedures are known in the art.
  • a PBMC enrichment procedure is a ficoll density gradient centrifugation process that separates the main cell populations, such as lymphocytes, monocytes, granulocytes, and red blood cells, throughout a density gradient medium.
  • the aqueous medium includes ficoll, a hydrophilic polysaccharide that forms the high density solution. Layering of whole blood over or under a density medium without mixing of the two layers followed by centrifugation will disperse the cells according to their densities with the PBMC fraction forming a thin white layer at the interface between the plasma and the density gradient medium (see e.g., Panda and Ravindran (2013) Isolation of Human PBMCs. BioProtoc. Vol. 3(3)).
  • centripetal forces can be used to separate PBMCs from other blood components, in ficoll using the spinning force of a Sepax cell processing system.
  • apheresis for example leukapheresis
  • PBMCs can be used to isolate cells, such as PBMCs.
  • AMICUS RBCX Frsenius-Kabi
  • Trima Accel Tuumo BCT
  • Cells isolated by apheresis typically contain T cells, B cells, NK cells, monocytes, granulocytes, other nucleated white blood cells, red blood cells, and/or platelets.
  • the cells collected by apheresis can be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media, such as phosphate buffered saline (PBS) or wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations, for subsequent processing steps.
  • PBS phosphate buffered saline
  • the cells collected by apheresis can be genetically modified by any of the methods provided herein.
  • the cells collected by apheresis can be used to prepare any of the cell formulations provided herein.
  • the cells collected by apheresis can be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS.
  • leukopheresis can be used to isolate cells, such as lymphocytes.
  • a leukopak can be used in any embodiment that includes TNCs.
  • a huffy coat can be used in another PBMC enrichment method.
  • an automated leukapheresis collection system such as SPECTRA OPTIA® APHERESIS SYSTEM from Terumo BCT, Inc.
  • Lakewood, CO 80215, USA is used to separate the inflow of whole blood from the target PBMC fraction using high-speed centrifugation while typically returning the outflow material, such as plasma, red blood cells, and granulocytes, back to the donor, although this returning would be optional in methods provided herein. Further processing may be necessary to remove residual red blood cells and granulocytes. Both methods include a time intensive purification of the PBMCs, and the leukapheresis method requires the presence and participation of the patient during the PBMC enrichment step.
  • PBMCs are isolated using a Sepax or Sepax 2 cell processing system (BioSafe).
  • the PBMCs are isolated using a CliniMACS Prodigy cell processor (Miltenyi Biotec).
  • an automated apheresis separator is used which takes blood from the subject, passes the blood through an apparatus that sorts out a particular cell type (such as, for example, PBMCs), and returns the remainder back into the subject. Density gradient centrifugation can be performed after apheresis.
  • the PBMCs are isolated using a leukoreduction filter assembly.
  • magnetic bead activated cell sorting is then used for purifying a specific cell population from PBMCs, such as, for example, PBLs or a subset thereof, according to a cellular phenotype (i.e., positive selection), before they are used in a reaction mixture herein.
  • Other methods for purification can also be used, such as, for example, substrate adhesion, which utilizes a substrate that mimics the environment that a T cell encounters during recruitment, to purify T cells before adding them to a reaction mixture, or negative selection can be used, in which unwanted cells are targeted for removal with antibody complexes that target the unwanted cells for removal before a reaction mixture for a contacting step is formed.
  • red blood cell resetting can be used to remove red blood cells before forming a reaction mixture.
  • hematopoietic stem cells can be removed before a contacting step, and thus in these embodiments, are not present during the contacting step.
  • an ABC transporter inhibitor and/or substrate is not present before, during, or both before and during the contacting (i.e., not present in the reaction mixture in which contacting takes place) with or without optional incubating, or any step of the method.
  • lymphocytes are modified and in illustrative embodiments genetically modified and/or transduced with prior activation and/or stimulation and cultured ex vivo until a desired number of cells to be delivered are achieved.
  • lymphocytes are modified and in illustrative embodiments genetically modified and/or transduced without prior activation or stimulation, and/or without requiring prior activation or stimulation, whether in vivo, in vitro, or ex-vivo', and/or furthermore, in some embodiments, without ex vivo or in vitro activation or stimulation after an initial contacting with or without an optional incubation, or without requiring ex vivo or in vitro activation or stimulation after an initial contacting with or without an optional incubation.
  • the cell is activated during the contacting and is not activated at all or not activated for more than 15 minutes, 30 minutes, 1, 2, 4, or 8 hours before the contacting.
  • activation by elements that are not present on the retroviral particle surface is not required for modifying, genetically modifying, and/or transducing the cell. Accordingly, such activation or stimulation elements are not required other than on the retroviral particle, before, during, or after the contacting.
  • these illustrative embodiments that do not require pre-activation or stimulation provide the ability to rapidly perform in vitro experiments aimed at better understanding T cells and the biologicals mechanisms, therein.
  • lymphocytes e.g., NK cells and especially T cells
  • rPOC rapid point-of-care
  • some, most, at least 25%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 99%, or all of the lymphocytes are resting when they are combined with retroviral particles to form a reaction mixture, and typically are resting when they are contacted with retroviral viral particles in a reaction mixture.
  • lymphocytes can be contacted in the typically resting state they were in when present in the collected blood in vivo immediately before collection.
  • the T cells and/or NK cells consist of between 95 and 100% resting cells (Ki-67 ).
  • the T cell and/or NK cells that are contacted by replication incompetent recombinant retroviral particles include between 90, 91, 92, 93, 94, and 95% resting cells on the low end of the range and 96, 97, 98, 99, or 100% resting cells on the high end of the range.
  • the T cells and/or NK cells include naive cells.
  • the subembodiments in this paragraph are included in composition and method aspects for transducing lymphocytes in whole blood.
  • contact between the T cells and/or NK cells and the replication incompetent recombinant retroviral particles can facilitate transduction of the T cells and/or NK cells by the replication incompetent recombinant retroviral particles.
  • the replication incompetent recombinant retroviral particles identify and bind to T cells and/or NK cells and the T cells and NK cells are “modified” as the term is used herein.
  • genetic material from the replication incompetent recombinant retroviral particles enters the T cells and/or NK cells at which time the T cells and/or NK cells are “genetically modified” as the phrase is used herein. It is noteworthy that such process might occur hours or even days after the contacting is initiated, and even after non-associated retroviral particles are rinsed away.
  • the genetic material is typically integrated into the genomic DNA of the T cells and/or NK cells, at which time the T cells and/or NK cells are now “transduced” as the term is used herein.
  • cells can be modified, genetically modified, and/or transduced by recombinant vectors other than replication incompetent recombinant retroviral particles. Cells may also internalize and integrate genetic material into the genomic DNA of the T cells and/or NK cells after transfection, at which time the T cells and/or NK cells are now “stably transfected” as the term is used herein.
  • any method for modifying and/or genetically modifying lymphocytes is a method for transducing lymphocytes (e.g., T cells and/or NK cells). It is believed that by day 6 in vivo or ex vivo, after contacting is initiated, the vast majority of modified and genetically modified cells have been transduced. Methods of lentiviral transduction are known. Exemplary methods are described in, e.g., Wang et al. (2012) J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101: 1637-1644; Verhoeyen et al. (2009) Methods Mol Biol.
  • a transduced, or in some embodiments a stably transfected, T cell and/or NK cell includes progeny of ex vivo transduced cells that retain at least some of the nucleic acids or polynucleotides that are incorporated into the genome of a cell during the ex vivo transduction.
  • methods herein that recite “reintroducing” a transduced cell it will be understood that such cell is typically not in a transduced state when it is collected from the blood of a subject.
  • a T cell activation element in illustrative embodiments is present in the reaction mixture where initial contacting of a recombinant retrovirus and lymphocytes occurs.
  • T cell activation element can be in solution in the reaction mixture.
  • soluble anti-CD3 antibodies can be present in the reaction mixture during the contacting and optional incubation thereafter, at 25-200, 50-150, 75-125, or 100 ng/ml.
  • the soluble anti-CD3 antibodies are multivalent such as bivalent, tetravalent, or a higher order valency.
  • the T cell activation element is associated with the retroviral surface.
  • the T cell activation element can be any T cell activation element provided herein.
  • the T cell activation element can be anti-CD3, such as anti-CD3 scFv, or anti-CD3 scFvFc.
  • the replication incompetent recombinant retroviral particle can further include a T cell activation element, which in further illustrative examples is associated with the external side of the surface of the retrovirus.
  • the contacting step of a method for transducing and/or a method for modifying or genetically modifying lymphocytes in whole blood typically includes an initial step in which the retroviral particle, typically a population of retroviral particles, are brought into contact with blood cells, typically a population of blood cells that includes an anticoagulant and/or additional blood components other than PBMCs, that are not present after a PBMC enrichment procedure, while in suspension in a liquid buffer and/or media to form a transduction reaction mixture.
  • this contacting can be followed by an optional incubating period in this reaction mixture that includes the retroviral particles and the blood cells comprising lymphocytes (e.g., T cells and/or NK cells) in suspension.
  • lymphocytes e.g., T cells and/or NK cells
  • the reaction mixture can include at least one, two, three, four, five, or all additional blood components as disclosed herein, and in illustrative embodiments includes one or more anticoagulants.
  • the transduction reaction mixture in any of the aspects provided herein can be incubated at between 23 and 39 °C, and in some illustrative embodiments at 37 °C, in an optional incubation step after the initial contacting of retroviral particles and lymphocytes.
  • the transduction reaction can be carried out at 37-39 °C for faster fusion/transduction.
  • the contacting step is a cold contacting step as discussed elsewhere herein, with an optional incubating step.
  • the cold contacting step is performed at temperatures less than 37 °C, such as between 1 °C and 25 °C or 2 °C and 6 °C.
  • the optional incubating associated with the contacting step at these temperatures can be performed for any length of time discussed herein, for example in the Exemplary Embodiments section. In illustrative embodiments, the optional incubating associated with these temperatures is performed for 8 hours, 6 hours, 4 hours, 2 hours, and in illustrative embodiments 1 hour or less.
  • the contacting is carried out at a lower temperature, for example between 2 °C and 25 °C, referred to herein as cold contacting, and then retroviral particles that remain unassociated in suspension are removed from the reaction mixture, for example by washing the reaction mixture over a filter, such as a leukoreduction filter, that retains leukocytes including T cells and NK cells, but not free, unassociated viral particles.
  • a filter such as a leukoreduction filter, that retains leukocytes including T cells and NK cells, but not free, unassociated viral particles.
  • the cells and retroviral particles when brought into contact in the transduction reaction mixture can be immediately processed to remove the retroviral particles that remain free in suspension and not associated with cells, from the cells.
  • the cells in suspension and retroviral particles whether free in suspension or associated with the cells in suspension can be incubated for various lengths of time, as provided herein for a contacting step in a method provided herein.
  • a wash can be performed, regardless of whether such cells will be studied in vitro, ex vivo or introduced into a subject.
  • Such suspension can include allowing cells and retroviral particles to settle or causing such settling through application of a force, such as a centrifugal force, to the bottom of a vessel or chamber, as discussed in further detail herein.
  • a force such as a centrifugal force
  • such g force is lower than the g forces used successfully in spinoculation procedures.
  • the methods provided herein allow for rapid ex vivo processing of lymphocytes, and in certain illustrative embodiments, PBMCs, and in other illustrative embodiments, total nucleated cells (TNCs), without an ex vivo expansion step, fundamentally simplifying the delivery of adoptive cell therapies, for example by providing such point-of-care methods, and in some illustrative embodiments, in shorter periods of time (rapid point-of-care (rPOC)).
  • rPOC rapid point-of-care
  • Illustrative methods are disclosed herein for modifying lymphocytes, especially NK cells and in illustrative embodiments, T cells, that are much shorter and simpler than prior methods.
  • the contacting step in any method provided herein of transducing, genetically modifying, and/or modifying a PBMC or a lymphocyte, typically a T cell and/or an NK cell can be performed (or can occur) for any of the time periods provided in this specification, included, but not limited to those provided in the Exemplary Embodiments section.
  • said contacting can be for less than 24 hours, for example, less than 12 hours, less than 8 hours, less than 4 hours, less than 2 hours, less than 1 hour, less than 30 minutes or less than 15 minutes, but in each case there is at least an initial contacting step in which retroviral particles and cells come into contact in suspension in a transduction reaction mixture before retroviral particles that remain in suspension not associated with a cell, are separated from cells and typically discarded, as discussed in further detail herein.
  • contacting begins at the time that retroviral particles and lymphocytes are combined together, typically by adding a solution containing the retroviral particles into a solution containing lymphocytes (e.g., T cells and/or NK cells).
  • lymphocytes e.g., T cells and/or NK cells
  • the reaction mixture containing cells and recombinant nucleic acid vectors which in illustrative embodiments include nucleic acids that encode an anti-idiotype polypeptide, and in further illustrative embodiments are retroviral particles, in suspension for a specified time period without removing recombinant nucleic acid vectors (e.g., retroviral particles) that remain free in solution and not associated with cells.
  • This incubating is sometimes referred to herein as an optional incubation.
  • the contacting can be performed (or can occur) for between 15 minutes and 12 hours, between 15 minutes and 10 hours, or between 15 minutes and 8 hours, or any of the times included in the Exemplary Embodiments section.
  • a secondary incubation is performed by suspending cells after an optional wash step such that recombinant nucleic acid vectors, and in illustrative embodiments retroviral particles, that are not associated with a cell are washed away.
  • the secondary incubation is performed at temperatures between 32 °C and 42 °C, such as at 37 °C.
  • the optional secondary incubation can be performed for any length of time discussed herein. In illustrative embodiments, the optional secondary incubation is performed for 6 hours or less.
  • the contacting can be performed (or can occur) (where as indicated in general herein the low end of a selected range is less than the high end of the selected range) for between 30 seconds or 1, 2, 5, 10, 15, 30, or 45 minutes, or 1, 2, 3, 4, 5, 6, 7, or 8 hours on the low end of the range, and between 10 minutes, 15 minutes, 30 minutes, or 1, 2, 4, 6, 8, 10, 12, 18, 24, 36, 48, and 72 hours on the high end of the range.
  • the reaction mixture can be incubated for between 5 minutes on the low end of the range and 10, 15, or 30 minutes or 1, 2, 3, 4, 5, 6, 8, 10 or 12 hours on the high end of the range. In other embodiments, the reaction mixture can be incubated for between 15 minutes and 12 hours, 15 minutes and 10 hours, 15 minutes and 8 hours, 15 minutes and 6 hours, 15 minutes and 4 hours, 15 minutes and 2 hours, 15 minutes and 1 hour, 15 minutes and 45 minutes, or 15 minutes and 30 minutes.
  • the reaction mixture can be incubated for between 30 minutes and 12 hours, 30 minutes and 10 hours, 30 minutes and 8 hours, 30 minutes and 6 hours, 30 minutes and 4 hours, 30 minutes and 2 hours, 30 minutes and 1 hour, or 30 minutes and 45 minutes. In other embodiments, the reaction mixture can be incubated for between 1 hour and 12 hours, 1 hour and 8 hours, 1 hour and 4 hours, or Ihour and 2 hours. In another illustrative embodiment, the contacting is performed for between an initial contacting step only (without any further incubating in the reaction mixture including the retroviral particles free in suspension and cells in suspension) without any further incubation in the reaction mixture, or a 5 minute, 10 minute, 15 minute, 30 minute, or 1 hour incubation in the reaction mixture.
  • blood cells or a T cell and/or NK cell-containing fraction thereof in the reaction mixture are separated from retroviral particles that are not associated with such cells.
  • a PBMC enrichment procedure e.g., a Ficoll gradient in a Sepax unit
  • this can be performed using a PBMC enrichment procedure (e.g., a Ficoll gradient in a Sepax unit), or in certain illustrative embodiments provided herein, by filtering the reaction mixture over a leukocyte depletion filter set assembly, and then collecting the leukocytes, which include T cells and NK cells.
  • this can be performed by centrifugation of the reaction mixture at a relative centrifugal force less than 500 g, for example 400 g, or between 300 and 490 g, or 350 and 450 g.
  • centrifugation to separate retroviral particles from cells can be performed for example, for between 5 minutes and 15 minutes, or between 5 minutes and 10 minutes.
  • g force is typically lower than the g forces used successfully in spinoculation procedures.
  • a method provided herein in any aspect does not involve performing a spinoculation.
  • the cell or cells are not subjected to a spinoculation of at least 400 g, 500 g, 600 g, 700 g, or 800 g for at least 15 minutes.
  • the cell or cells are not subjected to a spinoculation of at least 800 g for at least 10, 15, 20, 25, 30, 35, 40, or 45 minutes.
  • spinoculation is included as part of a contacting step.
  • spinoculation when spinoculation is performed there is no additional incubating as part of the contacting, as the time of the spinoculation provides the incubation time of the optional incubation discussed above. In other embodiments, there is an additional incubation after the spinoculating of between 15 minutes and 4 hours, 15 minutes and 2 hours, or 15 minutes and 1 hour.
  • the spinoculation can be performed for example, for 30 minutes to 120 minutes, typically for at least 60 minutes, for example for 60 minutes to 180 minutes, or 60 minutes to 90 minutes.
  • the spinoculation is typically performed in a centrifuge with a relative centrifugal force of at least 800 g, and more typically at least 1200 g, for example between 800 g and 2400 g, 800 g and 1800 g, 1200 g and 2400 g, or 1200 g and 1800 g.
  • a relative centrifugal force typically at least 800 g, and more typically at least 1200 g, for example between 800 g and 2400 g, 800 g and 1800 g, 1200 g and 2400 g, or 1200 g and 1800 g.
  • such methods typically involve an additional step of resuspending the pelleted cells and retroviral particles, and then removing retroviral particles that are not associated with cells according to steps discussed above when spinoculation is not performed.
  • the contacting step including the optional incubation therein, and the spinoculation, in embodiments that include spinoculation, can be performed at between 4 °C and 42 °C or 20 °C and 37 °C. In certain illustrative embodiments, spinoculation is not performed and the contacting and associated optional incubation are carried out at 20-25 °C for 4 hours or less, 2 hours or less, 1 hour or less, 30 minutes or less, 15 minutes or less, or 15 minutes to 2 hours, 15 minutes to 1 hour, or 15 minutes to 30 minutes.
  • Methods of genetically modifying lymphocytes typically include insertion into the cell of a polynucleotide comprising one or more transcriptional units encoding any transgene, for example one, two, three or all of an anti-idiotype polypeptide, a cytokine a CAR , and a lymphoproliferative element, and in illustrative embodiments includes nucleic acids encoding the anti-idiotype polypeptide according to any of the embodiments provided herein.
  • lymphoproliferative element can be provided to support the shorter and more simplified methods provided herein, which can support expansion of modified, genetically modified, and/or transduced T cells and/or NK cells after the contacting and optional incubation. Accordingly, in exemplary embodiments of any methods provided herein, lymphoproliferative elements can be delivered from the genome of the retroviral particles inside genetically modified, and/or transduced T cells and/or NK cells, such that those cells have the characteristics of increased proliferation and/or survival disclosed in the Lymphoproliferative Elements section herein.
  • the genetically modified T cell or NK cell is capable of engraftment in vivo in mice and/or enrichment in vivo in mice for at least 7, 14, or 28 days.
  • mice may be treated or otherwise genetically modified so that any immunological differences between the genetically modified T cell and/or NK cell do not result in an immune response being elicited in the mice against any component of the lymphocyte transduced by the replication incompetent recombinant retroviral particle.
  • Media that can be included in a contacting step for example when the cells and retroviral particles are initially brought into contact, or in any aspects provided herein, during optional incubation periods with the reaction mixture thereafter that include retroviral particles and cells in suspension in the media, or media that can be used during cell culturing and/or during various wash steps in any aspects provided herein, can include base media such as commercially available media for ex vivo T cell and/or NK cell culture.
  • Non-limiting examples of such media include, X-VIVOTM 15 Chemically Defined, Serum-free Hematopoietic Cell Medium (Lonza) (2018 catalog numbers BE02-060F, BE02-00Q, BE-02- 061Q, 04-744Q, or 04-418Q), ImmunoCultTM-XF T Cell Expansion Medium (STEMCELL Technologies) (2018 catalog number 10981), PRIME-XV® T Cell Expansion XSFM (Irvine Scientific) (2018 catalog number 91141), AIM V® Medium CISTM (Therapeutic Grade) (Thermo Fisher Scientific (Referred to herein as “Thermo Fisher”), or CTSTM OptimizerTM media (Thermo Fisher) (2018 catalog numbers A10221-01 (basal media (bottle)), and A10484-02 (supplement), A10221-03 (basal media (bag)), A1048501 (basal media and supplement kit (bottle)) and, A1048503 (basal media and supplement
  • Such media can be a chemically defined, serum-free formulation manufactured in compliance with cGMP, as discussed herein for kit components.
  • the media can be xeno-free and complete.
  • the base media has been cleared by regulatory agencies for use in ex vivo cell processing, such as an FDA 510(k) cleared device.
  • the media is the basal media with or without the supplied T cell expansion supplement of 2018 catalog number A1048501 (CTSTM OpTmizerTM T Cell Expansion SFM, bottle format) or A1048503 (CTSTM OpTmizerTM T Cell Expansion SFM, bag format) both available from Thermo Fisher (Waltham, MA).
  • Additives such as human serum albumin, human AB+ serum, and/or serum derived from the subject can be added to the transduction reaction mixture.
  • Supportive cytokines can be added to the transduction reaction mixture, such as IL2, IL7, or IL 15, or those found in human sera.
  • dGTP can be added to the transduction reaction in certain embodiments.
  • the cells can be contacted with a retroviral particle without prior activation.
  • the retroviral particle can include, as non-limiting examples, nucleic acids that encode an anti-idiotype polypeptide.
  • the T cells and/or NK cells have not been incubated on a substrate that adheres to monocytes for more than 4 hours in one embodiment, or for more than 6 hours in another embodiment, or for more than 8 hours in another embodiment before the transduction.
  • the T cells and/or NK cells have been incubated overnight on an adherent substrate to remove monocytes before the transduction.
  • the method can include incubating the T cells and/or NK cells on an adherent substrate that binds monocytes for no more than 30 minutes, 1 hour, or 2 hours before the transduction.
  • the T cells and/or NK cells are exposed to no step of removing monocytes by an incubation on an adherent substrate before said transduction step.
  • the T cells and/or NK cells are not incubated with or exposed to a bovine serum, such as a cell culturing bovine serum, for example fetal bovine serum before or during a contacting step and/or a modifying and/or a genetically modifying and/or transduction step.
  • a bovine serum such as a cell culturing bovine serum, for example fetal bovine serum before or during a contacting step and/or a modifying and/or a genetically modifying and/or transduction step.
  • reaction mixtures formed in such methods, and modified, genetically modified, and/or transduced lymphocytes (e.g., T cells and/or NK cells) made by such methods can be contained within such a closed system.
  • a closed system is a cell processing system that is generally closed or fully closed to an environment, such as an environment within a room or even the environment within a hood, outside of the conduits such as tubes, and chambers, of the system in which cells are processed and/or transported.
  • Such closed system methods can be performed with commercially available devices. Different closed system devices can be used at different steps within a method and the cells can be transferred between these devices using tubing and connections such as welded, luer, spike, or clave ports to prevent exposure of the cells or media to the environment.
  • blood can be collected into an IV bag or syringe, optionally including an anticoagulant, and in some aspects, transferred to a Sepax 2 device (Biosafe) for PBMC enrichment and isolation.
  • whole blood can be filtered to collect leukocytes using a leukoreduction filter assembly.
  • the isolated PBMCs or isolated leukocytes can be transferred to a chamber of a G-Rex device for an optional activation, a transduction and optional expansion.
  • collected blood can be transduced in a blood bag, for example, the bag in which it was collected.
  • the cells can be harvested and collected into another bag using a Sepax 2 device.
  • the methods can be carried out in any device or combination of devices adapted for closed system T cell and/or NK cell production.
  • Non-limiting examples of such devices include G-Rex devices (Wilson Wolf), GatheRex (Wilson Wolf), Sepax 2 (Biosafe), WAVE Bioreactors (General Electric), a CultiLife Cell Culture bag (Takara), a PermaLife bag (OriGen), CliniMACS Prodigy (Miltenyi Biotec), and VueLife bags (Saint-Gobain).
  • the optional activating, the transducing and optional expanding can be performed in the same chamber or vessel in the closed system.
  • the chamber can be a chamber of a G-Rex device and PBMCs or leukocytes can be transferred to the chamber of the G-Rex device after they are enriched and isolated, and can remain in the same chamber of the G-Rex device until harvesting.
  • Methods provided herein can include transferring blood and cells therein and/or fractions thereof, as well as lymphocytes before or after they are contacted with retroviral particles, between vessels within a closed system, which thus is without environmental exposure.
  • Vessels used in the closed system can be a tube, bag, syringe, or other container.
  • the vessel is a vessel that is used in a research facility.
  • the vessel is a vessel used in commercial production.
  • the vessel can be a collection vessel used in a blood collection process.
  • Methods for modifying herein typically involve a contacting step wherein lymphocytes are contacted with a replication incompetent recombinant retroviral particle.
  • the contacting in some embodiments, can be performed in the vessel, for example, within a blood bag. Blood and various lymphocyte-containing fractions thereof, can be transferred from the vessel to another vessel (for example from a first vessel to a second vessel) within the closed system for the contacting.
  • the second vessel can be a cell processing compartment of a closed device, such as a G-Rex device.
  • genetically modified (e.g., transduced) cells can be transferred to a different vessel within the closed system (i.e., without exposure to the environment). Either before or after this transfer the cells are typically washed within the closed system to remove substantially all or all of the retroviral particles.
  • a process disclosed herein, from collection of blood, to contacting (e.g., transduction), optional incubating, and post-incubation isolation and optional washing, is performed for between 15 minutes, 30 minutes, or 1, 2, 3, or 4 hours on the low end of the range, and 4, 8, 10, or 12 hours on the high end of the range.
  • a T cell can include any of the embodiments of TIPs provided herein, including those that include one or more anti-idiotype polypeptide, lymphoproliferative element, CAR, pseudotyping element, control element, activation element, membrane-bound cyto
  • the retroviral particle is a lentiviral particle.
  • a method for modifying, genetically modifying, and/or transducing a PBMC or a lymphocyte, such as a T cell and/or NK cell can be performed in vitro or ex vivo.
  • a skilled artisan will recognize that details provided herein for transducing, genetically modifying, and/or modifying PBMCs or lymphocytes, such as T cell(s) and/or NK cell(s) can apply to any aspect that includes such step(s).
  • Introduction or reintroduction, also referred to herein as administration and readministration, of modified cells, such as modified lymphocytes and in illustrative embodiments genetically modified lymphocytes, or in some embodiments, RIPs into a subject in methods provided herein can be via any route known in the art.
  • Such introduction or reintroduction of genetically modified lymphocytes typically involves suspending i) modified and/or ii) genetically modified and/or iiia) transduced or iiib) transfected cells, in a delivery solution to form a cell formulation that can be introduced or reintroduced into a subject as discussed in further detail herein.
  • introduction of RIPs can involve suspension of the RIPs in a delivery solution to form a transducing formulation that can be introduced into a subject.
  • introduction or RIPS, lymphocytes or modified lymphocytes, or reintroduction for lymphocytes or modified lymphocytes can be delivery via infusion into a blood vessel of the subject.
  • RIPS or modified lymphocytes e.g., T cells and/or NK cells
  • Some administered cells are modified with a nucleic acid encoding a lymphoproliferative element.
  • a nucleic acid encoding a lymphoproliferative element is not limited by theory, in non-limiting illustrative methods, the delivery of a polynucleotide encoding a lymphoproliferative element, to a resting T cell and/or NK cell ex vivo, which can integrate into the genome of the T cell or NK cell, provides that cell with a driver for in vivo expansion without the need for lymphodepleting the host.
  • the subject is not exposed to a lymphodepleting agent within 1, 2, 3, 4, 5, 6, 7, 10, 14, 21, or 28 days, or within 1 month, 2 months, 3 months or 6 months of performing the contacting, during the contacting, and/or within 1, 2, 3, 4, 5, 6, 7, 10, 14, 21, or 28 days, or within 1 month, 2 months, 3 months or 6 months after the modified T cells and/or NK cells are reintroduced back into the subject.
  • methods provided herein can be performed without exposing the subject to a lymphodepleting agent during a step wherein a replication incompetent recombinant retroviral particle is in contact with resting T cells and/or resting NK cells of the subject and/or during the entire ex vivo method.
  • methods of expanding modified and in illustrative embodiments genetically modified T cells and/or NK cells in a subject in vivo is a feature of some embodiments of the present disclosure.
  • such methods involve ex-vivo propagation of modified cells, and in illustrative embodiments, such methods are ex vivo propagation-free or substantially propagation-free.
  • This entire method/process from blood draw from a subject to reintroduction of modified and in illustrative embodiments genetically modified lymphocytes into the subject after ex vivo transduction of T cells and/or NK cells can occur over a time period less than 48 hours, less than 36 hours, less than 24 hours, less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, 2 hours, or less than 2 hours.
  • introduction or reintroduction of the modified lymphocytes can be performed by intravenous injection, intraperitoneal administration, subcutaneous administration, intratumoral, or intramuscular administration.
  • the entire method/process from blood draw/collection from a subject to reintroduction of modified lymphocytes into the subject after ex vivo transduction of T cells and/or NK cells occurs over a time period between 1 hour and 12 hours, 2 hours and 8 hours, 1 hour and 3 hours, 2 hours and 4 hours, 2 hours and 6 hours, 4 hours and 12 hours, 4 hours and 24 hours, 8 hours and 24 hours, 8 hours and 36 hours, 8 hours and 48 hours, 12 hours and 24 hours, 12 hours and 36 hours, or 12 hours and 48 hours, or over a time period between 15, 30, 60, 90, 120, 180, and 240 minutes on the low end of the range, and 120, 180, and 240, 300, 360, 420, and 480 minutes on the high
  • the entire method/process from blood draw/collection from a subject to reintroduction of modified and in illustrative embodiments genetically modified lymphocytes into the subject after ex vivo transduction of T cells and/or NK cells occurs over a time period between 1, 2, 3, 4, 6, 8, 10, and 12 hours on the low end of the range, and 8, 9, 10, 11, 12, 14, 18, 24, 36, or 48 hours on the high end of the range.
  • the modified and genetically modified T cells and/or NK cells are separated from the nonassociated replication incompetent recombinant retroviral particles after the time period in which contact occurs.
  • illustrative embodiments of methods provided herein for modifying lymphocytes, and associated methods for performing adoptive cell therapy can be performed in significantly less time than prior methods, fundamental improvements in patient care and safety as well as product manufacturability are made possible. Therefore, such processes are expected to be favorable in the view of regulatory agencies responsible for approving such processes when carried out in vivo for therapeutic purposes.
  • the subject in non-limiting examples of any aspects provided herein that include a subject can remain in the same building (e.g., infusion clinic) or room as the instrument processing their blood or sample for the entire time that the sample is being processed before modified T cells and/or NK cells are reintroduced into the patient.
  • a subject remains within line of site and/or within 100, 50, 25, or 12 feet or arm’s distance of their blood or cells that are being processed, for the entire method/process from blood draw/collection from the subject to reintroduction of blood to the subject after ex vivo transduction of T cells and/or NK cells.
  • a subject remains awake and/or at least one person can continue to monitor the blood or cells of the subject that are being processed, throughout and/or continuously for the entire method/process from blood draw/collection from the subject to reintroduction of blood to the subject after ex vivo transduction of T cells and/or NK cells.
  • the entire method/process for adoptive cell therapy and/or for transducing resting T cells and/or NK cells from blood draw/collection from the subject to reintroduction of blood to the subject after ex vivo transduction of T cells and/or NK cells can be performed with continuous monitoring by a human.
  • the entire method/process from blood draw/collection from the subject to reintroduction of blood to the subject after ex vivo transduction of T cells and/or NK cells are blood cells incubated in a room that does not have a person present.
  • the entire method/process from blood draw/collection from the subject to reintroduction of blood to the subject after ex vivo transduction of T cells and/or NK cells is performed next to the subject and/or in the same room as the subject and/or next to the bed or chair of the subject.
  • sample identity mix-ups can be avoided, as well as long and expensive incubations over periods of days or weeks.
  • methods provided herein are readily adaptable to closed and automated blood processing systems, where a blood sample and its components that will be reintroduced into the subject, only make contact with disposable, single-use components.
  • Methods for modifying, genetically modifying, and/or transducing cells can be part of a method for delivering polynucleotides such as polynucleotide vectors to a subject, for delivering modified cells to a subject, or for performing adoptive cell therapy.
  • these methods steps of collecting blood from a subject, and returning modified, genetically modified, and/or transduced lymphocytes (e.g., T cells and/or NK cells) to the subject.
  • the present disclosure provides various treatment methods using a CAR, and in illustrative embodiments an anti-idiotype polypeptide, which can be a CAR that has an anti-idiotype extracellular recognition domain.
  • a CAR of the present disclosure when present in a T lymphocyte or an NK cell, can mediate cytotoxicity toward a target cell.
  • a CAR of the present disclosure binds to an antigen present on a target cell, thereby mediating killing of a target cell by a T lymphocyte or an NK cell genetically modified to produce the CAR.
  • the ASTR of the CAR binds to an antigen present on the surface of a target cell.
  • the present disclosure provides methods of killing, or inhibiting the growth of, a target cell, the method involving contacting a cytotoxic immune effector cell (e.g., a cytotoxic T cell, or an NK cell) that is genetically modified to produce a subject CAR, such that the T lymphocyte or NK cell recognizes an antigen present on the surface of a target cell, and mediates killing of the target cell.
  • a cytotoxic immune effector cell e.g., a cytotoxic T cell, or an NK cell
  • the target cell can be a cancer cell, for example, and autologous cell therapy methods herein, can be methods for treating cancer, in some illustrative embodiments.
  • the subject can be a an animal or human suspected of having cancer, or more typically, a subject that is known to have cancer.
  • genetically modified cells can be administered in combination with an anti-PDL-1 antibody or antibody mimetic.
  • DLBCL diffuse large B cell lymphoma
  • cells are introduced or reintroduced into the subject by infusion into a vein or artery, especially when neutrophils are not present in a preparation of lymphocytes that have been contacted with retroviral particles and are ready to be reintroduced, or by subcutaneous, intratumoral, or intramuscular administration, for embodiments where at least 1%, 2%, 3%, 4%, 5%, 7.5%, 10%, 15%, 20% or 25% of the cells, or between 1% and 90%, 1% and 75%, 1% and 50%, 1% and 25%, 1% and 20%, 1% and 10%, 5% and 90%, 5% and 75%, 5% and 50%, 5% and 25%, 5% and 20%, 5% and 10%, 10% and 90%, 10% and 75%, 10% and 50%, 10% and 25%, orl0% and 20%, of the cells in a cell formulation to be administered are neutrophils.
  • Such embodiments can include coadministration or sequential administration with hyaluronidase, as discussed in further detail herein.
  • the number of lymphocytes, and in illustrative embodiments modified T cells and/or NK cells, present in cell formulations provided herein and optionally reinfused or in illustrative embodiments, subcutaneously delivered into a subject can be between 1 x 10 3 , 2.5 x 10 3 , 5 x
  • the number of lymphocytes, and in illustrative embodiments modified T cells and/or NK cells, present in cell formulations herein and optionally reinfused or otherwise delivered into a subject can be between 1 x 10 4 , 2.5 x 10 4 , 5 x 10 4 , 1 x 10 5 , 2.5 x 10 5 , 5 x 10 5 , 1 x 10 6 , 2.5 x 10 6 , 5 x 10 6 , and 1 x 10 7 cells/kg on the low end of the range and 5 x 10 4 , 1 x 10 5 , 2.5 x 10 5 , 5 x 10 5 , 1 x 10 6 , 2.5 x 10 6 , 5 x 10 6 , 1 x 10 7 , 2.5 x 10 7 , 5 x 10 7 , 1 x 10 8 , 1 x 10 9 , and 1 x 10 10 cells/kg on the high end of the range.
  • the number of lymphocytes, and in illustrative embodiments T cells and/or NK cells present in cell formulations herein and optionally reinfused, or delivered intratumorally, intramuscularly, subcutaneously, or otherwise delivered into a subject can be between 5 x 10 5 , 1 x 10 6 , 2.5 x 10 6 , 5 x 10 6 , 1 x 10 7 , 2.5 x 10 7 , 5 x 10 7 , and 1 x IO 8 cells on the low end of the range and 2.5 x 10 6 , 5 x 10 6 , 1 x 10 7 , 2.5 x 10 7 , 5 x 10 7 , 1 x IO 8 , 2.5 x IO 8 , 5 x IO 8 , and 1 x 10 9 cells on the high end of the range.
  • the number of lymphocytes, and in illustrative embodiments T cells and/or NK cells, present in cell formulations herein and available for infusion, reinfusion, or other delivery means (e.g., subcutaneous delivery) into a 70 kg subject or patient is between 7 x 10 5 and 2.5 x 10 8 cells.
  • the number of lymphocytes, and in illustrative embodiments T cells and/or NK cells present in cell formulations herein and available for transduction is approximately 7 x 10 6 plus or minus 10%.
  • the cell can be an autologous cell or an allogeneic cell.
  • the allogeneic cell can be a genetically engineered allogeneic cell. Allogeneic cells, such as allogeneic T cells, and methods for genetically engineering allogeneic cells, are known in the art.
  • the allogeneic cell is a T cell
  • the T cell has been genetically engineered such that at least one component of the TCR complex is functionally impaired and/or is at least partially deleted.
  • the T cell has been genetically engineered such that the expression of at least one component of the TCR complex has been reduced or eliminated.
  • the allogeneic cell can be modified such that it is missing all or part of the B2 microglobulin gene.
  • allogeneic cells can include any of the lymphoproliferative elements and/or CLEs disclosed herein. The use of lymphoproliferative elements and CLEs can reduce the required number of cells and can facilitate cell manufacturing of T cells, NK cells, B cells, or stem cells.
  • the allogeneic cell can be an immortalized cell. In any of the aspects or embodiments herein that include an allogeneic cell, steps that include collecting blood or contacting a cell with a replication incompetent recombinant retroviral particle can be eliminated.
  • a T cell may have been previously genetically modified, and the genetically modified allogeneic CAR-T cell is administered to the subject without collecting blood from the subject.
  • the allogeneic cell is administered subcutaneously.
  • the allogeneic cell is administered intravenously.
  • the allogeneic cell is administered intraperitoneally.
  • lymphocytes e.g., T cells and/or NK cells
  • RIPs replication incompetent recombinant retroviral particles
  • the modified, genetically modified, and/or transduced lymphocyte (e.g., T cell and/or NK cell) or population thereof, or the RIPs in compositions provided herein without cells, such as GMP RIP compositions, are introduced or reintroduced into the subject.
  • Introduction or reintroduction of the modified and in illustrative embodiments genetically modified lymphocytes into a subject can be via any route known in the art.
  • introduction or reintroduction can be delivery via infusion into a blood vessel of the subject.
  • the modified, genetically modified, and/or transduced lymphocyte (e.g., T cell and/or NK cell) or population thereof undergo 4 or fewer cell divisions ex vivo prior to being introduced or reintroduced into the subject.
  • the lymphocyte(s) used in such a method are resting T cells and/or resting NK cells that are in contact with the replication incompetent recombinant retroviral particles for between 1 hour and 12 hours. In some embodiments, no more than 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, or 1 hour pass(es) between the time blood is collected from the subject and the time the modified and/or genetically modified T cells and/or NK cells are formulated for delivery and/or are reintroduced into the subject. In some embodiments, all steps after the blood are collected and before the blood is reintroduced, are performed in a closed system in which a person monitors the closed system throughout the processing.
  • the modified and in illustrative embodiments genetically modified T cells and/or NK cells are introduced back, reintroduced, reinfused or otherwise delivered into the subject without additional ex vivo manipulation, such as stimulation and/or activation of T cells and/or NKs.
  • ex vivo manipulation is used for stimulation/activation of T cells and/or NK cells and for expansion of genetically modified T cells and/or NK cells prior to introducing the genetically modified T cells and/or NK cells into the subject.
  • this generally takes days or weeks and requires a subject to return to a clinic for a blood infusion days or weeks after an initial blood draw.
  • T cells and/or NK cells are not stimulated ex vivo by exposure to anti-CD3 alone or anti-CD3 in combination with co-stimulation by, for example, anti-CD28, either in solution or attached to a solid support such as, for example, beads coated with anti-CD3/anti-CD28, prior to contacting the T cells and/or NK cells with the replication incompetent recombinant retroviral particles.
  • anti-CD3 alone or anti-CD3 in combination with co-stimulation by, for example, anti-CD28, either in solution or attached to a solid support such as, for example, beads coated with anti-CD3/anti-CD28, prior to contacting the T cells and/or NK cells with the replication incompetent recombinant retroviral particles.
  • modified and in illustrative embodiments genetically modified T cells and/or NK cells are not expanded ex vivo, or only expanded for a small number of cell divisions (e.g., 1, 2, 3, 4, or 5 rounds of cell division), but are rather expanded, or predominantly expanded, in vivo, i.e., within the subject.
  • no additional media is added to allow for further expansion of the cells.
  • no cell manufacturing of the primary blood lymphocytes (PBLs) occurs while the PBLs are contacted with the replication incompetent recombinant retroviral particles.
  • no cell manufacturing of the PBLs occurs while the PBLs are ex vivo.
  • subjects are lymphodepleted prior to reinfusion with genetically modified T cells and or NK cells.
  • patients or subjects are not lymphodepleted prior to infusion or reinfusion with modified and/or genetically modified T cells and or NK cells.
  • embodiments of the methods and compositions disclosed herein can be used on pre-activated or pre-stimulated T cells and/or NK cells as well.
  • T cells and/or NK cells can be stimulated ex vivo by exposure to anti-CD3 with or without anti-CD28 solid supports prior to contacting the T cells and/or NK cells with the replication incompetent recombinant retroviral particles.
  • the T cells and/or NK cells can be exposed to anti-CD3/anti-CD28 solid supports for less than 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, or 24 hours, including no exposure, before the T cells and/or NK cells are contacted the replication incompetent recombinant retroviral particles.
  • the T cells and/or NK cells can be exposed to anti-CD3/anti-CD28 solid supports for less than 1, 2, 3, 4, 6, or 8 hours before the T cells and/or NK cells are contacted the replication incompetent recombinant retroviral particles.
  • any cell in a cell mixture, cell formulation, or reaction mixture that is useful in adoptive cell therapy can be enriched prior to formulation for delivery.
  • the desired cells can be enriched by positive selection prior to being contacted with a recombinant nucleic acid vector, such as a replication incompetent retroviral particle.
  • the desired cells can be enriched by positive selection after the cell mixture, cell formulation, or reaction mixture is contacted with a recombinant nucleic acid vector, such as a replication incompetent retroviral particle.
  • enriching the one or more cell populations can be performed at the same time as any of the methods of genetic modification disclosed herein, and in illustrative embodiments genetic modification with a replication incompetent retroviral particle.
  • Mononuclear cells such as PBMCs
  • TNCs can be isolated from a more complex cell mixture such as whole blood by density-gradient centrifugation or reverse perfusion of a leukoreduction filter assembly, respectively, as described in more detail herein.
  • the desired cells can have specific cell lineages, such as NK cells, T cells, and/or T cell subsets including naive, effector, memory, suppressor T-cells, and/or regulatory T cells and can be enriched through the selection of cells expressing one or more surface molecules.
  • the one or more surface molecules can include CD4, CD8, CD16, CD25, CD27, CD28, CD44, CD45RA, CD45RO, CD56, CD62L, CCR7, KIRs, FoxP3, and/or TCR components such as CD3.
  • Methods using beads conjugated to antibodies directed to one or more surface molecules can be used to enrich for the desired cells using magnetic, density, and size-based separation.
  • the replication incompetent recombinant retroviral particles used to contact T cells and/or NK cells have a polynucleotide or nucleic acid having one or more transcriptional units that encode one or more engineered signaling polypeptides.
  • an engineered signaling polypeptide includes any combination of an extracellular domain (e.g., an antigen-specific targeting region or ASTR), a stalk and a transmembrane domain, combined with one or more intracellular activating domains, optionally one or more modulatory domains (such as a co-stimulatory domain), and optionally one or more T cell survival motifs.
  • At least one, two, or all of the engineered signaling polypeptides is a chimeric antigen receptor (CAR) or a lymphoproliferative element (LE) such as a chimeric lymphoproliferative element (CLE).
  • at least one, two, or all of the engineered signaling polypeptides is an engineered T cell receptor (TCR).
  • TCR T cell receptor
  • when two signaling polypeptides are utilized one encodes a lymphoproliferative element and the other encodes a chimeric antigen receptor (CAR) that includes an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activating domain.
  • ASTR antigen-specific targeting region
  • engineered signaling polypeptides For any domain of an engineered signaling polypeptide disclosed herein, exemplary sequences can be found in WO2019/055946, incorporated herein in its entirety by reference. A skilled artisan will recognize that such engineered polypeptides can also be referred to as recombinant polypeptides.
  • the engineered signaling polypeptides such as CARs, engineered TCRs, LEs, and CLEs provided herein, are typically transgenes with respect to lymphocytes, especially T cells and NK cells, and most especially T cells and/or NK cells that are engineered using methods and compositions provided herein, to express such signaling polypeptides.
  • an engineered signaling polypeptide includes an extracellular domain that is a member of a specific binding pair.
  • the extracellular domain can be the extracellular domain of a cytokine receptor, or a mutant thereof, or a hormone receptor, or a mutant thereof.
  • Such mutant extracellular domains in some embodiments have been reported to be constitutively active when expressed at least in some cell types.
  • such extracellular and transmembrane domains do not include a ligand binding region. It is believed that such domains do not bind a ligand when present in an engineered signaling polypeptide and expressed in B cells, T cells, and/or NK cells.
  • Mutations in such receptor mutants can occur in the extracellular juxtamembrane region.
  • a mutation in at least some extracellular domains (and some extracellular- transmembrane domains) of engineered signaling polypeptides provided herein are responsible for signaling of the engineered signaling polypeptide in the absence of ligand, by bringing activating chains together that are not normally together.
  • extracellular domains that comprise mutations in extracellular domains can be found, for example, in the Lymphoproliferative Element section herein.
  • the extracellular domain comprises a dimerizing motif.
  • the dimerizing motif comprises a leucine zipper.
  • the leucine zipper is from a jun polypeptide, for example c-jun. Further embodiments regarding extracellular domains that comprise a dimerizing motif can be found, for example, in the Lymphoproliferative Element section herein.
  • the extracellular domain is an antigen-specific targeting region (ASTR), sometimes called an antigen binding domain herein.
  • ASTR antigen-specific targeting region
  • Specific binding pairs include, but are not limited to, antigen-antibody binding pairs; ligand-receptor binding pairs; and the like.
  • a member of a specific binding pair suitable for use in an engineered signaling polypeptide of the present disclosure includes an ASTR that is an antibody, an antigen, a ligand, a receptor binding domain of a ligand, a receptor, a ligand binding domain of a receptor, and an alternative non-antibody scaffold, also referred to herein as an antibody mimetic.
  • the ASTR can be a suitable antibody mimetic.
  • the antibody mimetic can be an affibody, an afflilin, an affimer, an affitin, an alphabody, an alphamab, an anticalin, a peptide aptamer, an armadillo repeat protein, an atrimer, an avimer (also known as avidity multimer), a C-type lectin domain, a cysteine- knot miniprotein, a cyclic peptide, a cytotoxic T-lymphocyte associated protein-4, a DARPin (Designed Ankyrin Repeat Protein), a fibrinogen domain, a fibronectin binding domain (FN3 domain) (e.g., adnectin or monobody), a fynomer, a knottin, a Kunitz domain peptide, a nanofitin, a leucine -rich repeat domain,
  • An ASTR suitable for use in an engineered signaling polypeptide of the present disclosure can be any antigen-binding polypeptide.
  • the ASTR is an antibody such as a full-length antibody, a single-chain antibody, a Fab fragment, a Fab' fragment, a (Fab')2 fragment, a Fv fragment, and a divalent single-chain antibody or a diabody.
  • the ASTR is a single chain Fv (scFv).
  • the heavy chain is positioned N-terminal of the light chain in the engineered signaling polypeptide.
  • the light chain is positioned N-terminal of the heavy chain in the engineered signaling polypeptide.
  • the heavy and light chains can be separated by a linker as discussed in more detail herein.
  • the heavy or light chain can be at the N-terminus of the engineered signaling polypeptide and is typically C-terminal of another domain, such as a signal sequence or peptide.
  • cAb VHH camelid antibody variable domains
  • IgNAR VH shk antibody variable domains
  • sdAb VH single domain antibody variable domains
  • camelized antibody variable domains are suitable for use with the engineered signaling polypeptides and methods using the engineered signaling polypeptides of the present disclosure.
  • T cell receptor (TCR) based recognition domains are suitable for use with the engineered signaling polypeptides and methods using the engineered signaling polypeptides of the present disclosure.
  • TCR T cell receptor
  • Naturally-occurring T cell receptors include an a-subunit and a [3-subunit, separately produced by unique recombination events in a T cell's genome.
  • Libraries of TCRs may be screened for their selectivity to a target antigen, for example, any of the antigens disclosed herein. Screens of natural and/or engineered TCRs can identify TCRs with high avidities and/or reactivities towards a target antigen.
  • TCRs can be selected, cloned, and a polynucleotide encoding such a TCR can be included in a replication incompetent recombinant retroviral particle to genetically modify a lymphocyte, or in illustrative embodiments, T cell or NK cell, such that the lymphocyte expresses the engineered TCR.
  • the TCR can be a single chain TCR (scTv, single chain two-domain TCR containing VaV[3).
  • a CAR include CARs having extracellular domains engineered to co-opt the endogenous TCR signaling complex and CD3Z signaling pathway.
  • a chimeric antigen receptor ASTR is fused to one of the endogenous TCR complex chains (e.g., TCR alpha, CD3E etc.) to promote incorporation into the TCR complex and signaling through the endogenous CD3Z chains.
  • a CAR contains a first scFv or protein that binds to the TCR complex and a second scFv or protein that binds to the target antigen (e.g., tumor antigen).
  • the TCR can be a single chain TCR (scTv, single chain two-domain TCR containing VaVP).
  • scFv single chain two-domain TCR containing VaVP
  • scFv single chain two-domain TCR containing VaVP
  • the ASTR can be multispecific, e.g., bispecific antibodies.
  • Multispecific antibodies have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for one target antigen and the other is for another target antigen.
  • bispecific antibodies may bind to two different epitopes of a target antigen. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express a target antigen. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.
  • any of the CARs of the current disclosure can be multispecific.
  • one of the ASTRs on a multispecific, e.g., bispecific can be an anti-idiotype extracellular recognition domain as disclosed elsewhere herein.
  • An ASTR suitable for use in an engineered signaling polypeptide of the present disclosure, or an engineered TCR can have a variety of antigen-binding specificities.
  • the antigen-binding domain is specific for an epitope present in an antigen that is expressed by (synthesized by) a target cell.
  • the target cell is a cancer cell associated antigen.
  • the cancer cell associated antigen can be an antigen associated with, e.g., a breast cancer cell, a B cell lymphoma cell, as a diffuse large B cell lymphoma (DLBCL) cell, a Hodgkin lymphoma cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma, a lung cancer cell (e.g., a small cell lung cancer cell), a lymphoma cell, a non-Hodgkin B- cell lymphoma (B-NHL) cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma cell, a lung cancer cell (e.g., a small cell lung cancer cell), a melanoma cell, a leukemia cell, a chronic myelogenous leukemia (CML) cell, a chronic lymphocytic leukemia (CLL) cell, an acute myelogenous leukemia (AML) cell, an acute lympho
  • a cancer cell associated antigen may also be expressed by a non-cancerous cell.
  • the cancer cell is a PDL-1 positive cancer cell.
  • the cancer cell is a PDL-1 positive DLBCL cell.
  • the cancer cell is a PDL-1 negative cell.
  • the cancer cell is a PDL-1 negative DLBCL cell.
  • the antigen can be a tumor-associated antigen or a tumor-specific antigen.
  • the tumor- associated antigen or tumor-specific antigen is Axl, ROR1, ROR2, Her2 (ERBB2), prostate stem cell antigen (PSCA), PSMA (prostate-specific membrane antigen), B cell maturation antigen (BCMA), alphafetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, chromogranin, protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-Dl, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma
  • EMA epithelial membrane protein
  • ETA epithelial tumor antigen
  • tyrosinase
  • the ASTR or recombinant TCR can recognize, bind to, or otherwise interact with the idiotype of a target antibody or antibody mimetic, as disclosed elsewhere herein.
  • the anti-idiotype polypeptide is a CAR or TCR wherein the ASTR is the anti-idiotype external recognition domain of an anti-idiotype polypeptide.
  • the ASTR can be any of the extracellular recognition domains of an anti-idiotype polypeptide disclosed herein.
  • the ASTR can recognize the idiotcetuximab, muromonab-CD3, efalizumab, tositumomab-il31, nebacumab, edrecolomab, catumaxomab, daclizumab, olaratumab, abciximab, rituximab, basiliximab, palivizumab, infliximab, trastuzumab, adalimumab, ibritumomab tiuxetan, omalizumab, bevacizumab, natalizumab, panitumumab, ranibizumab, eculizumab, certolizumab pegol, ustekinumab, canakinumab, golimumab, ofatumumab, tocilizumab, denosumab, belimumab, ipilimumab, brent
  • a member of a specific binding pair suitable for use in an engineered signaling polypeptide is an ASTR that is a ligand for a receptor.
  • Ligands include, but are not limited to, hormones (e.g., erythropoietin, growth hormone, leptin, etc.); cytokines (e.g., interferons, interleukins, certain hormones, etc.); growth factors (e.g., heregulin; vascular endothelial growth factor (VEGF); and the like); an integrin-binding peptide (e.g., a peptide comprising the sequence Arg-Gly-Asp (SEQ ID NO:1)); and the like.
  • hormones e.g., erythropoietin, growth hormone, leptin, etc.
  • cytokines e.g., interferons, interleukins, certain hormones, etc.
  • growth factors e.g., heregulin;
  • the engineered signaling polypeptide can be activated in the presence of a second member of the specific binding pair, where the second member of the specific binding pair is a receptor for the ligand.
  • the second member of the specific binding pair can be a VEGF receptor, including a soluble VEGF receptor.
  • the member of a specific binding pair that is included in an engineered signaling polypeptide is an ASTR that is a receptor, e.g., a receptor for a ligand, a co-receptor, etc.
  • the receptor can be a ligand-binding fragment of a receptor.
  • Suitable receptors include, but are not limited to, a growth factor receptor (e.g., a VEGF receptor); a killer cell lectin-like receptor subfamily K, member 1 (NKG2D) polypeptide (receptor for MICA, MICB, and ULB6); a cytokine receptor (e.g., an IL-13 receptor; an IL-2 receptor; etc.); CD27; a natural cytotoxicity receptor (NCR) (e.g., NKP30 (NCR3/CD337) polypeptide (receptor for HLA-B -associated transcript 3 (BAT3) and B7-H6); etc.); etc.
  • a growth factor receptor e.g., a VEGF receptor
  • a killer cell lectin-like receptor subfamily K, member 1 (NKG2D) polypeptide receptor for MICA, MICB, and ULB6
  • a cytokine receptor e.g., an IL-13 receptor; an IL-2 receptor; etc
  • the ASTR can be directed to an intermediate protein that links the ASTR with a target molecule expressed on a target cell.
  • the intermediate protein may be endogenously expressed or introduced exogenously and may be natural, engineered, or chemically modified.
  • the ASTR can be an anti-tag ASTR such that at least one tagged intermediate, typically an antibody-tag conjugate, is included between a tag recognized by the ASTR and a target molecule, typically a protein target, expressed on a target cell. Accordingly, in such embodiments, the ASTR binds a tag and the tag is conjugated to an antibody directed against an antigen on a target cell, such as a cancer cell.
  • Non-limiting examples of tags include fluorescein isothiocyanate (FITC), streptavidin, biotin, histidine, dinitrophenol, peridinin chlorophyll protein complex, green fluorescent protein, phycoerythrin (PE), horse radish peroxidase, palmitoylation, nitrosylation, alkaline phosphatase, glucose oxidase, and maltose binding protein.
  • FITC fluorescein isothiocyanate
  • streptavidin biotin
  • biotin histidine
  • dinitrophenol dinitrophenol
  • peridinin chlorophyll protein complex green fluorescent protein
  • PE phycoerythrin
  • horse radish peroxidase palmitoylation
  • nitrosylation alkaline phosphatase
  • glucose oxidase glucose oxidase
  • maltose binding protein a binds the tag.
  • the engineered signaling polypeptide includes a stalk which is located in the portion of the engineered signaling polypeptide lying outside the cell and interposed between the ASTR and the transmembrane domain.
  • the stalk has at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to a wild-type CD8 stalk region (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG AVHTRGLDFA (SEQ ID NO:2), has at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to a wild-type CD28 stalk region (FCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO:3)), or has at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to a wild-type immunoglobulin heavy chain stalk region.
  • the stalk employed allows the antigen-specific
  • the stalk region can have a length of from 4 to 250 amino acids, 10 to 250 amino acids, 4 to 200 amino acids, 4 to 100 amino acids, or 4 to 75 amino acids.
  • the stalk can have a length of from e.g., from 4 aa to 10 aa, from about 10 aa to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 40 aa, or from about 40 aa to about 50 aa.
  • the stalk separates the anti-id ERD from a cell membrane to which it is attached, at a great enough distance to permit binding of the anti-id ERD to its target antibody when they come into contact.
  • the stalk is a means for effectively separating an anti-id ERD from a cell membrane to which it is attached to permit binding of the anti-id ERD to its target antibody when they come into contact with each other.
  • the stalk of an engineered signaling polypeptide includes at least one cysteine.
  • the stalk can include the sequence Cys-Pro-Pro-Cys (SEQ ID NO:4). If present, a cysteine in the stalk of a first engineered signaling polypeptide can be available to form a disulfide bond with a stalk in a second engineered signaling polypeptide.
  • Stalks can include immunoglobulin hinge region amino acid sequences that are known in the art; see, e.g., Tan et al. (1990) Proc. Natl. Acad. Sci. USA 87:162; and Huck et al. (1986) Nucl. Acids Res. 14:1779.
  • an immunoglobulin hinge region can include a domain with at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids of any of the following amino acid sequences: DKTHT (SEQ ID NO:5); CPPC (SEQ ID NO:4); CPEPKSCDTPPPCPR (SEQ ID NO:6) (see, e.g., Glaser et al. (2005) J. Biol. Chem.
  • ELKTPLGDTTHT SEQ ID NO:7
  • KSCDKTHTCP SEQ ID NO:8
  • KCCVDCP SEQ ID NO:9
  • KYGPPCP SEQ ID NO: 10
  • EPKSCDKTHTCPPCP SEQ ID NO: 11
  • ERKCCVECPPCP SEQ ID NO: 12
  • ELKTPLGDTTHTCPRCP SEQ ID NO: 13
  • SPNMVPHAHHAQ SEQ ID NO: 14
  • the stalk can include a hinge region with an amino acid sequence of a human IgGl, IgG2, IgG3, or IgG4, hinge region.
  • the stalk can include one or more amino acid substitutions and/or insertions and/or deletions compared to a wild-type (naturally-occurring) hinge region.
  • His229 of human IgG 1 hinge can be substituted with Tyr, so that the stalk includes the sequence EPKSCDKTYTCPPCP (SEQ ID NO:15), (see, e.g., Yan et al. (2012) J. Biol. Chem. 287:5891).
  • the stalk can include an amino acid sequence derived from human CD8; e.g., the stalk can include the amino acid sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 16), or a variant thereof.
  • An engineered signaling polypeptide of the present disclosure can include transmembrane domains for insertion into a eukaryotic cell membrane.
  • the transmembrane domain can be interposed between the ASTR and the co-stimulatory domain.
  • the transmembrane domain can be interposed between the stalk and the co-stimulatory domain, such that the chimeric antigen receptor includes, in order from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus): an ASTR; a stalk; a transmembrane domain; and an activating domain.
  • any transmembrane (TM) domain that provides for insertion of a polypeptide into the cell membrane of a eukaryotic (e.g., mammalian) cell is suitable for use in aspects and embodiments disclosed herein.
  • the TM domain for any aspect provided herein that includes a CAR can include a transmembrane domain from BAFFR, C3Z, CEACAM1, CD2, CD3A, CD3B, CD3D, CD3E, CD3G, CD3Z, CD4, CD5, CD7, CD8A, CD8B, CD9, CD11A, CD11B, CD11C, CD11D, CD27, CD16, CD18, CD19, CD22, CD28, CD29, CD33, CD37, CD40, CD45, CD49A, CD49D, CD49F, CD64, CD79A, CD79B, CD80, CD84, CD86, CD96 (Tactile), CD100 (SEMA4D), CD103, C134, CD137,
  • TM domains suitable for any of the aspects or embodiments provided herein, include a domain with at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids of any of the following TM domains or combined stalk and TM domains: a) CD8 alpha TM (SEQ ID NO:17); b) CD8 beta TM (SEQ ID NO:18); c) CD4 stalk (SEQ ID NO: 19); d) CD3Z TM (SEQ ID NO:20); e) CD28 TM (SEQ ID NO:21); f) CD134 (0X40) TM: (SEQ ID NO:22); g) CD7 TM (SEQ ID NO:23); h) CD8 stalk and TM (SEQ ID NO:24); and i) CD28 stalk and TM (SEQ ID NO:25).
  • a transmembrane domain of an aspect of the invention can have at least 80%, 90%, or 95% or can have 100% sequence identity to the SEQ ID NO: 17 transmembrane domain, or can have 100% sequence identity to any of the transmembrane domains from the following genes respectively: the CD8 beta transmembrane domain, the CD4 transmembrane domain, the CD3 zeta transmembrane domain, the CD28 transmembrane domain, the CD 134 transmembrane domain, or the CD7 transmembrane domain.
  • Intracellular activating domains suitable for use in an engineered signaling polypeptide of the present disclosure when activated typically induce the production of one or more cytokines; increase cell death; and/or increase proliferation of CD8 + T cells, CD4 + T cells, NKT cells, y5 T cells, and/or neutrophils.
  • Activating domains can also be referred to as activation domains herein.
  • Activating domains can be used in CARs or in lymphoproliferative elements provided herein.
  • the intracellular activating domain includes at least one (e.g., one, two, three, four, five, six, etc.) IT AM motifs as described below.
  • an intracellular activating domain of an aspect of the invention can have at least 80%, 90%, or 95% or can have 100% sequence identity to the CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP12, FCER1G, FCGR2A, FCGR2C, DAP10/CD28, ZAP70, NKp30 (B7-H6), NKG2D, NKp44, NKp46, FcR gamma (FCER1G), FcR beta (FCER1B), FcgammaRI, FcgammaRIIA, FcgammaRIIC, FcgammaRIIIA, and FcRL5 domains as described below.
  • Intracellular activating domains suitable for use in an engineered signaling polypeptide of the present disclosure include immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides.
  • ITAM immunoreceptor tyrosine-based activation motif
  • An IT AM motif is YX1X2L/I, where Xi and X2 are independently any amino acid.
  • the intracellular activating domain of an engineered signaling polypeptide includes 1, 2, 3, 4, or 5 IT AM motifs.
  • an IT AM motif is repeated twice in an intracellular activating domain, where the first and second instances of the IT AM motif are separated from one another by 6 to 8 amino acids, e.g., (YXiX2L/I)(X3) n (YXiX2L/I), where n is an integer from 6 to 8, and each of the 6-8 X3 can be any amino acid.
  • the intracellular activating domain of an engineered signaling polypeptide includes 3 IT AM motifs.
  • a suitable intracellular activating domain can be an IT AM motif-containing portion that is derived from a polypeptide that contains an IT AM motif.
  • a suitable intracellular activating domain can be an IT AM motif-containing domain from any IT AM motif-containing protein.
  • a suitable intracellular activating domain need not contain the entire sequence of the entire protein from which it is derived.
  • IT AM motif-containing polypeptides include, but are not limited to: CD3Z (CD3 zeta); CD3D (CD3 delta); CD3E (CD3 epsilon); CD3G (CD3 gamma); CD79A (antigen receptor complex-associated protein alpha chain); CD79B (antigen receptor complex-associated protein beta chain) DAP12; and FCER1G (Fc epsilon receptor I gamma chain).
  • an intracellular activating domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all the amino acids in the following IT AM motif-containing polypeptides or to a contiguous stretch of from about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, or from about 150 aa to about 160 aa, of any of the following IT AM motif-containing polypeptides: CD3 zeta chain (also known as CD3Z, T cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.)
  • T cell surface glycoprotein CD3 gamma chain also known as CD3G, T cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc. with exemplary sequences:
  • CD79A also known as B-cell antigen receptor complex-associated protein alpha chain
  • CD79a antigen immunoglobulin-associated alpha
  • MB-1 membrane glycoprotein Ig-alpha
  • membrane-bound immunoglobulin-associated protein surface IgM-associated protein; etc.
  • Intracellular activating domains suitable for use in an engineered signaling polypeptide of the present disclosure include a DAP10/CD28 type signaling chain.
  • An example of a DAP10 signaling chain is the amino acid SEQ ID NO:50.
  • a suitable intracellular activating domain includes a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in SEQ ID NO:50.
  • a CD28 signaling chain is the amino acid sequence is SEQ ID NO:51.
  • a suitable intracellular domain includes a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids of SEQ ID NO:51.
  • Intracellular activating domains suitable for use in an engineered signaling polypeptide of the present disclosure include a ZAP70 polypeptide
  • a suitable intracellular activating domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in the following sequences or to a contiguous stretch of from about 300 amino acids to about 400 amino acids, from about 400 amino acids to about 500 amino acids, or from about 500 amino acids to 619 amino acids, of SEQ ID NO:52.
  • Modulatory domains can change the effect of the intracellular activating domain in the engineered signaling polypeptide, including enhancing or dampening the downstream effects of the activating domain or changing the nature of the response.
  • Modulatory domains suitable for use in an engineered signaling polypeptide of the present disclosure include co-stimulatory domains.
  • a modulatory domain suitable for inclusion in the engineered signaling polypeptide can have a length of from about 30 amino acids to about 70 amino acids (aa), e.g., a modulatory domain can have a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70 aa.
  • modulatory domain can have a length of from about 70 aa to about 100 aa, from about 100 aa to about 200 aa, or greater than 200 aa.
  • Co-stimulatory domains typically enhance and/or change the nature of the response to an activation domain.
  • Co-stimulatory domains suitable for use in an engineered signaling polypeptide of the present disclosure are generally polypeptides derived from receptors.
  • costimulatory domains homodimerize.
  • a subject co-stimulatory domain can be an intracellular portion of a transmembrane protein (i.e., the co-stimulatory domain can be derived from a transmembrane protein).
  • any of the CAR provided herein can include a costimulatory domain.
  • the co-stimulatory domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids or an intracellular domain of 4-1BB (CD137), B7-H3, B7-HCDR3, BAFFR, BTLA, C100 (SEMA4D), CD2, CD4, CD7, CD8A, CD8B, CD11A, CD11B, CD11C, CD11D, CD18, CD19, CD27, CD28, CD28 deleted for Lek binding (ICA), CD29, CD30, CD40, CD49A, CD49D, CD49F, CD69, CD84, CD96 (Tactile), CD103, CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (Ly9), a ligand that specifically binds with CD83, CDS,
  • a co-stimulatory domain suitable for inclusion in an engineered signaling polypeptide can have a length of from about 30 amino acids to about 70 amino acids (aa), e.g., a co-stimulatory domain can have a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70 aa.
  • the co-stimulatory domain can have a length of from about 70 aa to about 100 aa, from about 100 aa to about 200 aa, or greater than 200 aa.
  • a co-stimulatory domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all the amino acids or from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70 aa, from about 70 aa to about 75 aa, from about 75 aa to about 80 aa, from about 80 aa to about 85 aa, from about 85 aa to about 90 aa, from about 90 aa to about 95 aa, from about 95 aa to about 100
  • 0X40 contains a p85 PI3K binding motif at residues 34-57 and a TRAF binding motif at residues 76-102, each of SEQ ID NO: 296 (of Table 1).
  • the costimulatory domain can include the p85 PI3K binding motif of 0X40.
  • the costimulatory domain can include the TRAF binding motif of 0X40.
  • Lysines corresponding to amino acids 17 and 41 of SEQ ID NO: 296 are potentially negative regulatory sites that function as parts of ubiquitin targeting motifs. In some embodiments, one or both of these Lysines in the costimulatory domain of 0X40 are mutated Arginines or another amino acid.
  • the engineered signaling polypeptide includes a linker between any two adjacent domains.
  • a linker can be between the transmembrane domain and the first costimulatory domain.
  • the ASTR can be an antibody and a linker can be between the heavy chain and the light chain.
  • a linker can be between the ASTR and the transmembrane domain and a co-stimulatory domain.
  • a linker can be between the co-stimulatory domain and the intracellular activating domain of the second polypeptide.
  • the linker can be between the ASTR and the intracellular signaling domain.
  • the linker peptide may have any of a variety of amino acid sequences. Proteins can be joined by a spacer peptide, generally of a flexible nature, although other chemical linkages are not excluded.
  • a linker can be a peptide of between about 1 and about 100 amino acids in length, or between about 1 and about 25 amino acids in length. These linkers can be produced by using synthetic, linker-encoding oligonucleotides to couple the proteins. Peptide linkers with a degree of flexibility can be used.
  • the linking peptides may have virtually any amino acid sequence, bearing in mind that suitable linkers will have a sequence that results in a generally flexible peptide. The use of small amino acids, such as glycine and alanine, are of use in creating a flexible peptide. The creation of such sequences is routine to those of skill in the art.
  • Suitable linkers can be readily selected and can be of any of a suitable of different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.
  • Exemplary flexible linkers include glycine polymers (G) n , glycine-serine polymers (including, for example, (GS) n , (GSGGS) n , (GGS) n , (GGGS) n , and (GGGGS) n where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are of interest since both of these amino acids are relatively unstructured, and therefore may serve as a neutral tether between components.
  • Glycine polymers are of particular interest since glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)).
  • Exemplary flexible linkers include, but are not limited GGGGSGGGGS (SEQ ID NO:674), GGGGSGGGGSGGGGS (SEQ ID NO:63), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:372), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:675), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:64), GGSSRSS (SEQ ID NO:673), GGGGSGGGSGGGGS (SEQ ID NO:65), GGSG (SEQ ID NO:66), GGSGG (SEQ ID NO:67), GSGSG (SEQ ID NO:68), GSGGG (SEQ ID NO:69), GGGSG (SEQ ID NO:70), GSSSG (SEQ ID N0:71), and the like.
  • the ordinarily skilled artisan will recognize that design of a peptide conjugated to any elements described above can include linkers that are all or partially flexible, such that the linker can include a flexible linker
  • a polynucleotide provided by the replication incompetent recombinant retroviral particles has one or more transcriptional units that encode certain combinations of the one or more engineered signaling polypeptides.
  • modified and in illustrative embodiments genetically modified T cells include the combinations of the one or more engineered signaling polypeptides after transduction of T cells by the replication incompetent recombinant retroviral particles. It will be understood that the reference of a first polypeptide, a second polypeptide, a third polypeptide, etc. is for convenience and elements on a “first polypeptide” and those on a “second polypeptide” means that the elements are on different polypeptides that are referenced as first or second for reference and convention only, typically in further elements or steps to that specific polypeptide.
  • the first engineered signaling polypeptide includes an extracellular antigen binding domain, which is capable of binding an antigen, and an intracellular signaling domain. In other embodiments, the first engineered signaling polypeptide also includes a T cell survival motif and/or a transmembrane domain. In some embodiments, the first engineered signaling polypeptide does not include a co-stimulatory domain, while in other embodiments, the first engineered signaling polypeptide does include a co-stimulatory domain.
  • a second engineered signaling polypeptide includes a lymphoproliferative gene product and optionally an extracellular antigen binding domain.
  • the second engineered signaling polypeptide also includes one or more of the following: a T cell survival motif, an intracellular signaling domain, and one or more co-stimulatory domains.
  • at least one is a CAR.
  • the one or more engineered signaling polypeptides are expressed under a T cell specific promoter or a general promoter under the same transcript wherein in the transcript, nucleic acids encoding the engineered signaling polypeptides are separated by nucleic acids that encode one or more internal ribosomal entry sites (IREs) or one or more protease cleavage peptides.
  • IREs internal ribosomal entry sites
  • the polynucleotide encodes two engineered signaling polypeptides wherein the first engineered signaling polypeptide includes a first extracellular antigen binding domain, which is capable of binding to a first antigen, and a first intracellular signaling domain but not a costimulatory domain, and the second polypeptide includes a second extracellular antigen binding domain, which is capable of binding VEGF, and a second intracellular signaling domain, such as for example, the signaling domain of a co-stimulatory molecule.
  • the first antigen is PSCA, PSMA, or BCMA.
  • the first extracellular antigen binding domain comprises an antibody or fragment thereof (e.g., scFv), e.g., an antibody or fragment thereof specific to PSCA, PSMA, or BCMA.
  • the second extracellular antigen binding domain that binds VEGF is a receptor for VEGF, i.e., VEGFR.
  • the VEGFR is VEGFR1, VEGFR2, or VEGFR3. In a certain embodiment, the VEGFR is VEGFR2.
  • the polynucleotide encodes two engineered signaling polypeptides wherein the first engineered signaling polypeptide includes an extracellular tumor antigen binding domain and a CD3 ⁇ signaling domain, and the second engineered signaling polypeptide includes an antigenbinding domain, wherein the antigen is an angiogenic or vasculogenic factor, and one or more costimulatory molecule signaling domains.
  • the angiogenic factor can be, e.g., VEGF.
  • the one or more costimulatory molecule signaling motifs can comprise, e.g., co-stimulatory signaling domains from each of CD27, CD28, 0X40, ICOS, and 4-1BB.
  • the polynucleotide encodes two engineered signaling polypeptides wherein the first engineered signaling polypeptide includes an extracellular tumor antigen-binding domain and a CD3 ⁇ signaling domain, the second polypeptide comprises an antigen-binding domain, which is capable of binding to VEGF, and co-stimulatory signaling domains from each of CD27, CD28, 0X40, ICOS, and 4- IBB.
  • the first signaling polypeptide or second signaling polypeptide also has a T cell survival motif.
  • the T cell survival motif is, or is derived from, an intracellular signaling domain of IL-7 receptor (IL-7R), an intracellular signaling domain of IL- 12 receptor, an intracellular signaling domain of IL- 15 receptor, an intracellular signaling domain of IL-21 receptor, or an intracellular signaling domain of transforming growth factor [3 (TGF[3) receptor or the TGF[3 decoy receptor (TGF-[3 — dominant-negative receptor II (DNRII)).
  • IL-7R IL-7 receptor
  • TGF[3 transforming growth factor [3
  • TGF-[3 decoy receptor TGF-[3 — dominant-negative receptor II
  • the polynucleotide encodes two engineered signaling polypeptides wherein the first engineered signaling polypeptide includes an extracellular tumor antigen-binding domain and a CD3 ⁇ signaling domain, and the second engineered signaling polypeptide includes an antigen-binding domain, which is capable of binding to VEGF, an IL-7 receptor intracellular T cell survival motif, and co-stimulatory signaling domains from each of CD27, CD28, 0X40, ICOS, and 4- 1BB.
  • more than two signaling polypeptides are encoded by the polynucleotide.
  • only one of the engineered signaling polypeptides includes an antigen binding domain that binds to a tumor-associated antigen or a tumor-specific antigen; each of the remainder of the engineered signaling polypeptides comprises an antigen binding domain that binds to an antigen that is not a tumor-associated antigen or a tumor-specific antigen.
  • two or more of the engineered signaling polypeptides include antigen binding domains that bind to one or more tumor- associated antigens or tumor-specific antigens, wherein at least one of the engineered signaling polypeptides comprises an antigen binding domain that does not bind to a tumor-associated antigen or a tumor-specific antigen.
  • the antigen can be a tumor- associated antigen or a tumor-specific antigen.
  • the tumor-associated antigen or tumor-specific antigen is Axl, ROR1, ROR2, Her2 (ERBB2), prostate stem cell antigen (PSCA), PSMA (prostate-specific membrane antigen), B cell maturation antigen (BCMA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, chromogranin, protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-Dl, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA),
  • the first engineered signaling polypeptide includes a first extracellular antigen binding domain that binds a first antigen, and a first intracellular signaling domain; and a second engineered signaling polypeptide includes a second extracellular antigen binding domain that binds a second antigen, or a receptor that binds the second antigen; and a second intracellular signaling domain, wherein the second engineered signaling polypeptide does not comprise a co-stimulatory domain.
  • the first antigen-binding domain and the second antigen-binding domain are independently an antigen-binding portion of a receptor or an antigen-binding portion of an antibody.
  • first antigen binding domain or the second antigen binding domain are scFv antibody fragments.
  • first engineered signaling polypeptide and/or the second engineered signaling polypeptide additionally comprises a transmembrane domain.
  • the first engineered signaling polypeptide or the second engineered signaling polypeptide comprises a T cell survival motif, e.g., any of the T cell survival motifs described herein.
  • the first engineered signaling polypeptide includes a first extracellular antigen binding domain that binds HER2 and the second engineered signaling polypeptide includes a second extracellular antigen binding domain that binds MUC-1.
  • the second extracellular antigen binding domain of the second engineered signaling polypeptide binds an interleukin.
  • the second extracellular antigen binding domain of the second engineered signaling polypeptide binds a damage associated molecular pattern molecule (DAMP; also known as an alarmin).
  • DAMP is a heat shock protein, chromatin-associated protein high mobility group box 1 (HMGB1), S100A8 (also known as MRP8, or calgranulin A), S100A9 (also known as MRP14, or calgranulin B), serum amyloid A (SAA), deoxyribonucleic acid, adenosine triphosphate, uric acid, or heparin sulfate.
  • HMGB1 chromatin-associated protein high mobility group box 1
  • S100A8 also known as MRP8, or calgranulin A
  • S100A9 also known as MRP14, or calgranulin B
  • SAA serum amyloid A
  • said second antigen is an antigen on an antibody that binds to an antigen presented by a tumor cell.
  • signal transduction activation through the second engineered signaling polypeptide is non-antigenic, but is associated with hypoxia.
  • hypoxia is induced by activation of hypoxia-inducible factor-la (HIF-la), HIF-1[3, HIF-2a, HIF-2[3, HIF-3a, or HIF-3[3.
  • HIF-la hypoxia-inducible factor-la
  • expression of the one or more engineered signaling polypeptides is regulated by a control element, which is disclosed in more detail herein.
  • the engineered signaling polypeptides can further include one or more additional polypeptide domains, where such domains include, but are not limited to, a signal sequence; an epitope tag; an affinity domain; and a polypeptide whose presence or activity can be detected (detectable marker), for example by an antibody assay or because it is a polypeptide that produces a detectable signal.
  • Non- limiting examples of additional domains for any of the aspects or embodiments provided herein include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the following sequences as described below: a signal sequence, an epitope tag, an affinity domain, or a polypeptide that produces a detectable signal.
  • Signal sequences that are suitable for use in a subject CAR include any eukaryotic signal sequence, including a naturally-occurring signal sequence, a synthetic (e.g., man-made) signal sequence, etc.
  • the signal sequence can be the CD8 signal sequence MALPVTALLLPLALLLHAARP (SEQ ID NO:72).
  • Suitable epitope tags include, but are not limited to, hemagglutinin (HA; e.g., YPYDVPDYA; SEQ ID NO:73); FLAG (e.g., DYKDDDDK; SEQ ID NO:74); c-myc (e.g., EQKLISEEDL; SEQ ID NO:75), and the like.
  • Affinity domains include peptide sequences that can interact with a binding partner, e.g., such as one immobilized on a solid support, useful for identification or purification.
  • DNA sequences encoding multiple consecutive single amino acids, such as histidine, when fused to the expressed protein, may be used for one-step purification of the recombinant protein by high affinity binding to a resin column, such as nickel sepharose.
  • affinity domains include His5 (HHHHH; SEQ ID NO:76), HisX6 (HHHHHH; SEQ ID NO:77), c-myc (EQKLISEEDL; SEQ ID NO:75), Flag (DYKDDDDK; SEQ ID NO:74), Strep Tag (WSHPQFEK; SEQ ID NO:78), hemagglutinin, e.g., HA Tag (YPYDVPDYA; SEQ ID NO:73), GST, thioredoxin, cellulose binding domain, RYIRS (SEQ ID NO:79), Phe-His-His-Thr (SEQ ID NO: 80), chitin binding domain, S-peptide, T7 peptide, SH2 domain, C-end RNA tag, WEAAAREACCRECCARA (SEQ ID NO:81), metal binding domains, e.g., zinc binding domains or calcium binding domains such as those from calcium-binding proteins, e.g., calmodulin, troponin C, calc
  • Suitable detectable signal-producing proteins include, e.g., fluorescent proteins; enzymes that catalyze a reaction that generates a detectable signal as a product; and the like.
  • Suitable fluorescent proteins include, but are not limited to, green fluorescent protein (GFP) or variants thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2, t-dimer2(12), mRFPl, pocilloporin, Renilla GFP, Monster GFP, paGFP
  • fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrapel, mRaspberry, mGrape2, mPlum (Shaner et al. (2005) Nat. Methods 2:905-909), and the like. Any of a variety of fluorescent and colored proteins from Anthozoan species, as described in, e.g., Matz et al. (1999) Nature Biotechnol. 17:969-973, is suitable for use.
  • Suitable enzymes include, but are not limited to, horse radish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-N- acetylglucosaminidase, [3-glucuronidase, invertase, Xanthine Oxidase, firefly luciferase, glucose oxidase (GO), and the like.
  • HRP horse radish peroxidase
  • AP alkaline phosphatase
  • GAL beta-galactosidase
  • glucose-6-phosphate dehydrogenase beta-N- acetylglucosaminidase
  • [3-glucuronidase invertase
  • Xanthine Oxidase firefly luciferase
  • glucose oxidase GO
  • an engineered signaling polypeptide is a chimeric antigen receptor (CAR) or a polynucleotide encoding a CAR, which, for simplicity, is referred to herein as “CAR.”
  • a CAR of the present disclosure includes: a) at least one antigen-specific targeting region (ASTR); b) a transmembrane domain; and c) an intracellular activating domain.
  • the antigen-specific targeting region of the CAR is an scFv portion of an antibody to the target antigen.
  • the intracellular activating domain is from CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP12, FCER1G, FCGR2A, FCGR2C, DAP10/CD28, or ZAP70, and some further illustrative embodiments, from CD3z.
  • the CAR further comprises a co-stimulatory domain, for example any of the co-stimulatory domains provided above in the Modulatory Domains section, and in further illustrative embodiments the co-stimulatory domain is the intracellular co-stimulatory domain of 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM.
  • the CAR includes any of the transmembrane domains listed in the Transmembrane Domain section above.
  • a CAR of the present disclosure can be present in the plasma membrane of a eukaryotic cell, e.g., a mammalian cell, where suitable mammalian cells include, but are not limited to, a cytotoxic cell, a T lymphocyte, a stem cell, a progeny of a stem cell, a progenitor cell, a progeny of a progenitor cell, and an NK cell, an NK-T cell, and a macrophage.
  • a CAR of the present disclosure is active in the presence of one or more target antigens that, in certain conditions, binds the ASTR.
  • the target antigen is the second member of the specific binding pair.
  • the target antigen of the specific binding pair can be a soluble (e.g., not bound to a cell) factor; a factor present on the surface of a cell such as a target cell; a factor presented on a solid surface; a factor present in a lipid bilayer; and the like.
  • the antigen can be a soluble (e.g., not bound to a cell) antigen; an antigen present on the surface of a cell such as a target cell; an antigen presented on a solid surface; an antigen present in a lipid bilayer; and the like.
  • the ASTR of a CAR is expressed as a separate polypeptide from the intracellular signaling domain.
  • one or both of the polypeptides can include any of the transmembrane domains disclosed herein.
  • one or both of the polypeptides can include a heterologous signal sequence and/or a heterologous membrane attachment sequence.
  • the heterologous membrane attachment sequence is a GPI anchor attachment sequence.
  • a CAR of the present disclosure when present in the plasma membrane of a eukaryotic cell, and when activated by the one or more target antigens, increases expression of at least one nucleic acid in the cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10- fold, compared with the level of transcription of the nucleic acid in the absence of the one or more target antigens.
  • the CAR of the present disclosure can include an immunoreceptor tyrosine-based activation motif (IT AM) -containing intracellular signaling polypeptide.
  • IT AM immunoreceptor tyrosine-based activation motif
  • a CAR of the present disclosure when present in the plasma membrane of a eukaryotic cell, and when activated by one or more target antigens, can, in some instances, result in increased production of one or more cytokines by the cell.
  • a CAR of the present disclosure when present in the plasma membrane of a eukaryotic cell, and when activated by the one or more target antigens, can increase production of a cytokine by the cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10- fold, compared with the amount of cytokine produced by the cell in the absence of the one or more target antigens.
  • Cytokines whose production can be increased include, but are not limited to interferon gamma (IFN-y), tumor necrosis factor-alpha (TNF-a), IL-2, IL-15, IL-12, IL-4, IL-5, IL-10; a chemokine; a growth factor; and the like.
  • IFN-y interferon gamma
  • TNF-a tumor necrosis factor-alpha
  • IL-2 tumor necrosis factor-alpha
  • IL-15 IL-12
  • IL-4 IL-5
  • IL-10 a chemokine
  • chemokine a growth factor
  • a CAR of the present disclosure when present in the plasma membrane of a eukaryotic cell, and when activated by one or more target antigens, can result in both an increase in transcription of a nucleic acid in the cell and an increase in production of a cytokine by the cell.
  • a CAR of the present disclosure when present in the plasma membrane of a eukaryotic cell, and when activated by one or more target antigens, results in cytotoxic activity by the cell toward a target cell that expresses on its cell surface an antigen to which the antigen-binding domain of the first polypeptide of the CAR binds.
  • a CAR of the present disclosure when present in the plasma membrane of the cell, and when activated by the one or more target antigens, increases cytotoxic activity of the cell toward a target cell that expresses on its cell surface the one or more target antigens.
  • a CAR of the present disclosure when present in the plasma membrane of the cell, and when activated by the one or more target antigens, increases cytotoxic activity of the cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10-fold, compared to the cytotoxic activity of the cell in the absence of the one or more target antigens.
  • a CAR of the present disclosure when present in the plasma membrane of a eukaryotic cell, and when activated by one or more target antigens, can result in other CAR activation related events such as proliferation and expansion (either due to increased cellular division or anti- apoptotic responses).
  • a CAR of the present disclosure when present in the plasma membrane of a eukaryotic cell, and when activated by one or more target antigens, can result in other CAR activation related events such as intracellular signaling modulation, cellular differentiation, or cell death.
  • CARs of the present disclosure are microenvironment restricted. This property is typically the result of the microenvironment restricted nature of the ASTR domain of the CAR.
  • CARs of the present disclosure can have a lower binding affinity or, in illustrative embodiments, can have a higher binding affinity to one or more target antigens under a condition(s) in a microenvironment than under a condition in a normal physiological environment.
  • CARs provided herein comprise a co-stimulatory domain in addition to an intracellular activating domain, wherein the co-stimulatory domain is any of the intracellular signaling domains provided herein for lymphoproliferative elements (LEs), such as, for example, intracellular domains of CLEs.
  • Ls lymphoproliferative elements
  • the co-stimulatory domains of CARs herein are first intracellular domains (P3 domains) identified herein for CLEs or P4 domains that are shown as effective intracellular signaling domains of CLEs herein in the absence of a P3 domain.
  • co-stimulatory domains of CARs can comprise both a P3 and a P4 intracellular signaling domain identified herein for CLEs.
  • Certain illustrative subembodiments include especially effective P3 and P4 partner intracellular signaling domains as identified herein for CLEs.
  • the co-stimulatory domain is other than an ITAM-containing intracellular domain of a CAR either as part of the co-stimulatory domain, or in further illustrative embodiments as the only co-stimulatory domain.
  • the co-stimulatory domain of a CAR can be any intracellular signaling domain in Table 1 provided herein. Active fragments of any of the intracellular domains in Table 1 can be a co-stimulatory domain of a CAR.
  • the ASTR of the CAR comprises an scFv.
  • these CARs comprise an intracellular activating domain that in illustrative embodiments is a CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP12, FCER1G, FCGR2A, FCGR2C.
  • DAP10/CD28, or ZAP70 intracellular activating domain or in further illustrative embodiments is a CD3z intracellular activating domain.
  • the co-stimulatory domain of a CAR can comprise an intracellular domain or a functional signaling fragment thereof that includes a signaling domain from CSF2RB, CRLF2, CSF2RA, CSF3R, EPOR, GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL5RA, IL6R, IL6ST, IL7RA, IL9R, IL10RA, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17RB, IL17RC, IL17RD, IL18R1, IL18RAP, IL20RA, IL20RB, IL21R, IL22RA1, IL23R, IL27RA,
  • the co-stimulatory domain of a CAR can include an intracellular domain or a functional signaling fragment thereof that includes a signaling domain from CSF2RB, CRLF2, CSF2RA, CSF3R, EPOR, GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL5RA, IL6R, IL6ST, IL9R, IL10RA, IL10RB, IL11RA, IL13RA1, IL13RA2, IL17RB, IL17RC, IL17RD, IL18R1, IL18RAP, IL20RA, IL20RB, IL22RA1, IL31RA, LEPR, LIFR, LMP1, MPL, MyD88, OSMR, or PRLR.
  • the co-stimulatory domain of a CAR can include an intracellular domain or a functional fragment thereof that includes a signaling domain from CSF2RB, CSF2RA, CSF3R, EPOR, IFNGR1, IFNGR2, IL1R1, IL1RAP, IL1RL1, IL2RA, IL2RG, IL5RA, IL6R, IL9R, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA2, IL15RA, IL17RD, IL21R, IL23R, IL27RA, IL31RA, LEPR, MPL, MyD88, or OSMR.
  • the co-stimulatory domain of a CAR can include an intracellular domain or a fragment thereof that includes a signaling domain from CSF2RB, CSF2RA, CSF3R, EPOR, IFNGR1, IFNGR2, IL1R1, IL1RAP, IL1RL1, IL2RA, IL2RG, IL5RA, IL6R, IL9R, IL10RB, IL11RA, IL13RA2, IL17RD, IL31RA, LEPR, MPL, MyD88, or OSMR.
  • the co-stimulatory domain of a CAR can include an intracellular domain or a functional signaling fragment thereof that includes a signaling domain from CSF2RB, CSF3R, IFNAR1, IFNGR1, IL2RB, IL2RG, IL6ST, IL10RA, IL12RB2, IL17RC, IL17RE, IL18R1, IL27RA, IL31RA, MPL, MyD88, OSMR, or PRLR.
  • the co-stimulatory domain of a CAR can include an intracellular domain or a functional signaling fragment thereof that includes a signaling domain from CSF2RB, CSF3R, IFNGR1, IL2RB, IL2RG, IL6ST, IL10RA, IL17RE, IL31RA, MPL, or MyD88.
  • the co-stimulatory domain of a CAR can include an intracellular domain or a fragment thereof that includes a signaling domain from CSF3R, IL6ST, IL27RA, MPL, and MyD88.
  • the intracellular activating domain of the CAR is derived from CD3z.
  • TCRs T Cell Receptors
  • T Cell Receptors recognize specific protein fragments derived from intracellular as well as extracellular proteins. When proteins are broken into peptide fragments, they are presented on the cell surface with another protein called major histocompatibility complex, or MHC, which is called the HLA (human leukocyte antigen) complex in humans.
  • MHC major histocompatibility complex
  • HLA human leukocyte antigen
  • Such combinations are formed by dimerization between members of dimerizing subtypes, such as an a TCR subunit and a [3 TCR subunit, a y TCR subunit and a 5 TCR subunit, and for pre-TCRs, a pTa subunit and a [3 TCR subunit.
  • a set of TCR subunits dimerize and recognize a target peptide fragment presented in the context of an MHC.
  • the pre-TCR is expressed only on the surface of immature a[3 T cells while the a[3 TCR is expressed on the surface of mature a[3 T cells and NK T cells, and ySTCR is expressed on the surface of yST cells.
  • a[3TCRs on the surface of a T cell recognize the peptide presented by MHO or MHCII and the a[3 TCR on the surface of NK T cells recognize lipid antigens presented by CD1.
  • ySTCRs can recognize MHC and MHC-like molecules, and can also recognize non-MHC molecules such as viral glycoproteins.
  • a[3TCRs and ySTCRs transmit activation signals through the CD3zeta chain that stimulate T cell proliferation and cytokine secretion.
  • TCR molecules belong to the immunoglobulin superfamily with its antigen-specific presence in the V region, where CDR3 has more variability than CDR1 and CDR2, directly determining the antigen binding specificity of the TCR.
  • CDR3 has more variability than CDR1 and CDR2 directly determining the antigen binding specificity of the TCR.
  • the CDR1 and CDR2 recognize and bind the sidewall of the MHC molecule antigen binding channel, and the CDR3 binds directly to the antigenic peptide.
  • Recombinant TCRs may thus be engineered that recognize a tumor-specific protein fragment presented on MHC.
  • Recombinant TCR such as those derived from human TCRa and TCR[3 pairs that recognize specific peptides with common HLAs can thus be generated with specificity to a tumor specific protein (Schmitt, TM et al., 2009).
  • the target of recombinant TCRs may be peptides derived from any of the antigen targets for CAR ASTRs provided herein, but are more commonly derived from intracellular tumor specific proteins such as oncofetal antigens, or mutated variants of normal intracellular proteins or other cancer specific neoepitopes.
  • the recombinant TCR binds to the idiotype of an antibody.
  • the TCR is an anti-idiotype polypeptide as disclosed in more detail herein.
  • Libraries of TCR subunits may be screened for their selectivity to a target antigen. Screens of natural and/or recombinant TCR subunits can identify sets of TCR subunits with high avidities and/or reactivities towards a target antigen. Members of such sets of TCR subunits can be selected and cloned to produce one or more polynucleotide encoding the TCR subunit.
  • Polynucleotides encoding such a set of TCR subunits can be included in a replication incompetent recombinant retroviral particle to genetically modify a lymphocyte, or in illustrative embodiments, a T cell or an NK cell, such that the lymphocyte expresses the recombinant TCR.
  • the CAR in any aspect or embodiment provided herein that includes a polynucleotide encoding a CAR or an engineered signaling polypeptide that is a CAR, the CAR can be replaced by a set of ySTCR chains, or in illustrative embodiments a[3TCR chains.
  • TCR chains that form a set may be co-expressed using a number of different techniques to co-express the two TCR chains as is disclosed herein for expressing two or more other engineered signaling polypeptides such as CARs and lymphoproliferative elements.
  • protease cleavage epitopes such as 2A protease, internal ribosomal entry sites (IRES), and separate promoters may be used.
  • each member of the set of TCR chains in illustrative embodiments a[3TCR chains, comprises a modified constant domain that promotes preferential pairing with each other.
  • each member of a set of TCR chains, in illustrative embodiments a[3TCR chains comprises a mouse constant domain from the same TCR chain type, or a constant domain from the same TCR chain subtype with enough sequences derived from a mouse constant domain from the same TCR chain subtype, such that dimerization of the set of TCR chains to each other is preferred over, or occurs to the exclusion of, dimerization with human TCR chains.
  • each member of a set of TCR chains in illustrative embodiments a[3TCR chains, comprises corresponding mutations in its constant domain, such that dimerization of the set of TCR chains to each other is preferred over, or occurs to the exclusion of, dimerization with TCR chains that have human constant domains.
  • dimerization in illustrative embodiments, is under physiological conditions.
  • the constant regions of the members of each of the one or more sets of TCR chains are swapped.
  • the a TCR subunit of the set has a [3 TCR constant region
  • the [3 TCR subunit of the set has a a TCR constant region.
  • an engineered signaling polypeptide is a lymphoproliferative element (LE) such as a chimeric lymphoproliferative element (CLE).
  • L lymphoproliferative element
  • CLE chimeric lymphoproliferative element
  • the LE comprises an extracellular domain, a transmembrane domain, and at least one intracellular signaling domain that drives proliferation, and in illustrative embodiments a second intracellular signaling domain.
  • the extracellular domains, transmembrane domains, and intracellular domains of LEs can vary in their respective amino acid lengths.
  • the overall length of the LE can be between 3 and 4000 amino acids, for example between 10 and 3000, 10 and 2000, 50 and 2000, 250 and 2000 amino acids, and, in illustrative embodiments between 50 and 1000, 100 and 1000 or 250 and 1000 amino acids.
  • the extracellular domain when present to form an extracellular and transmembrane domain, can be between 1 and 1000 amino acids, and is typically between 4 and 400, between 4 and 200, between 4 and 100, between 4 and 50, between 4 and 25, or between 4 and 20 amino acids.
  • the extracellular region is GGGS for an extracellular and transmembrane domain of this aspect of the invention.
  • the transmembrane domains, or transmembrane regions of extracellular and transmembrane domains can be between 10 and 250 amino acids, and are more typically at least 15 amino acids in length, and can be, for example, between 15 and 100, 15 and 75, 15 and 50, 15 and 40, or 15 and 30 amino acids in length.
  • the intracellular signaling domains can be, for example, between 10 and 1000, 10 and 750, 10 and 500, 10 and 250, or 10 and 100 amino acids.
  • the intracellular signaling domain can be at least 30, or between 30 and 500, 30 and 250, 30 and 150, 30 and 100, 50 and 500, 50 and 250, 50 and 150, or 50 and 100 amino acids.
  • an intracellular signaling domain for a particular gene is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to at least 10, 25, 30, 40, 50, or all the amino acids from a sequence of that intracellular signaling domain, such as a sequence provided herein for that intracellular domain, up to the size of the entire intracellular domain sequence, and can include for example, up to an additional 1, 2, 3, 4, 5, 10, 20, or 25 amino acids, provided that such sequence still is capable of providing any of the properties of LEs disclosed herein.
  • the lymphoproliferative element can include a first and/or second intracellular signaling domain.
  • the first and/or second intracellular signaling domain can include CD2, CD3D, CD3E, CD3G, CD4, CD8A, CD8B, CD27, mutated Delta Lek CD28, CD28, CD40, CD79A, CD79B, CRLF2, CSF2RB, CSF2RA, CSF3R, EPOR, FCER1G, FCGR2C, FCGRA2, GHR, ICOS, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R, IL6ST, IL7RA, IL9R, IL10RA, IL10RB, IL11RA, IL12RB1,
  • the first intracellular signaling domain can include MyD88, or a functional mutant and/or fragment thereof.
  • the first intracellular signaling domain can include MyD88, or a functional mutant and/or fragment thereof
  • the second intracellular signaling domain can include ICOS, TNFRSF4, or TNSFR18, or functional mutants and/or fragments thereof.
  • the first intracellular domain is MyD88 and the second intracellular domain is an ITAM-containing intracellular domain, for example, an intracellular domain from CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP12, FCER1G, FCGR2A, FCGR2C, DAP10/CD28, or ZAP70.
  • the second intracellular signaling domain can include TNFRSF18, or a functional mutant and/or fragment thereof.
  • the lymphoproliferative element can include a fusion of an extracellular domain and a transmembrane domain.
  • the fusion of an extracellular domain and a transmembrane domain can include eTAG IL7RA Ins PPCL (interleukin 7 receptor), Myc LMP1, LMP1, eTAG CRLF2, eTAG CSF2RB, eTAG CSF3R, eTAG EPOR, eTAG GHR, eTAG truncated after Fn F523C IL27RA, or eTAG truncated after Fn S505N MPL, or functional mutants and/or fragments thereof.
  • the lymphoproliferative element can include an extracellular domain.
  • the extracellular domain can include cell tag with 0, 1, 2, 3, or 4 additional alanines at the carboxy terminus.
  • the extracellular domain can include Myc or an eTAG with 0, 1, 2, 3, or 4 additional alanines at the carboxy terminus, or functional mutants and/or fragments thereof.
  • a lymphoproliferative element disclosed herein that includes a cell tag there is a corresponding embodiment that is identical but lacks the cell tag and optionally lacks any linker sequence that connected the cell tag to the lymphoproliferative element.
  • the lymphoproliferative element can include a transmembrane domain.
  • the transmembrane domain can include a transmembrane domain from BAFFR, C3Z, CEACAM1, CD2, CD3A, CD3B, CD3D, CD3E, CD3G, CD3Z, CD4, CD5, CD7, CD8A, CD8B, CD9, CD11A, CD11B, CD11C, CD11D, CD27, CD16, CD18, CD19, CD22, CD28, CD29, CD33, CD37, CD40, CD45, CD49A, CD49D, CD49F, CD64, CD79A, CD79B, CD80, CD84, CD86, CD96 (Tactile), CD100 (SEMA4D), CD103, C134, CD137, CD154, CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (Ly9), CD247, CRLF2, C
  • CLEs for use in any aspect or embodiment herein can include any CLE disclosed in WO2019/055946 (incorporated by reference herein, in its entirety), the vast majority of which were designed to be and are believed to be constitutively active typically because they constitutively activate a signaling pathway, typically through functional domains on their intracellular domains.
  • the constitutively active signaling pathways include activation of a Jak pathway, a Stat pathway, or Jak/Stat pathways including Jakl, Jak2, Jak3, and Tyk2 and STATs such as STAT1, STAT2, STAT3, STAT4, STAT5, STAT6, and in illustrative embodiments, STAT3 and/or STAT5.
  • lymphoproliferative elements that comprises a means for activating any one or more of these pathways, and typically an intracellular domain that is a means for activating any one or more of these pathways.
  • lymphoproliferative elements comprise a means, such as an intracellular domain that is a means, for transmitting a signal that promotes proliferation of a T cell and/or NK cell, in illustrative embodiments when part of a dimerized lymphoproliferative element.
  • a CLE includes one or more STAT-activation domains.
  • a CLE includes two or more, three or more, four or more, five or more, or six or more STAT-activation domains.
  • At least one of the one or more STAT- activation domains is, or is derived from BLNK, IL2RG, EGFR, EpoR, GHR, IFNAR1, IFNAR2, IFNAR1/2, IFNLR1, IL10R1, IL12Rbl, IL12Rb2, IL21R, IL2Rb, IL2small, IL7R, IL7Ra, IL9R, IL15R, and IL21R, as are known in the art.
  • two or more STAT-activation domains are, or are derived from two or more different receptors.
  • the constitutively active signaling pathways include activation of a TRAF pathway through activation of TNF receptor associated factors such as TRAF3, TRAF4, TRAF7, and in illustrative embodiments TRAF1, TRAF2, TRAF5, and/or TRAF6.
  • lymphoproliferative elements for use in any of the kits, methods, uses, or compositions herein are constitutively active and comprise an intracellular signaling domain that activates a Jak/Stat pathway and/or a TRAF pathway.
  • the constitutively active signaling pathways include activation of PI3K pathways.
  • the constitutively active signaling pathways include activation of PEC pathways.
  • lymphoproliferative elements for use in any of the kits, methods, uses, or compositions herein are constitutively active and comprise an intracellular signaling domain that activates a Jak/Stat pathway a TRAF pathway, a PI3K pathway, and/or a PLC pathway.
  • the first intracellular signaling domain is positioned between the membrane associating motif, for example, a transmembrane domain, and the second intracellular domain.
  • the lymphoproliferative elements provided herein include one or more, or all of the binding domains, including those disclosed herein, responsible for signaling found in the corresponding lymphoproliferative element in nature.
  • the lymphoproliferative elements provided herein include one or more JAK binding domains.
  • the JAK- binding domain is, or is derived from, EPOR, GP130, PRLR, GHR, GCSFR, or TPOR/MPLR. JAK- binding domains from these proteins are known in the art and a skilled artisan will understand how to use them. For example, residues 273-338 of EpoR and residues 478-582 of TpoR are known to be JAK- binding domains.
  • a lymphoproliferative element herein is a transgenic B oxi -containing cytokine receptor that includes an intracellular domain of a cytokine receptor comprising a Boxl Janus kinase (JAK)-binding motif, optionally a Box2 JAK-binding motif, and a Signal Transducer and Activator of Transcription (STAT) binding motif comprising a tyrosine residue.
  • JAK Boxl Janus kinase
  • STAT Signal Transducer and Activator of Transcription
  • a lymphoproliferative element includes two or more JAK-binding motifs, for example three or more or four or more JAK-binding motifs, which in illustrative are the binding motifs found in natural versions of the corresponding lymphoproliferative element.
  • a lymphoproliferative element comprises an intracellular domain that is a means for transmitting a signal that promotes proliferation of a T cell and/or NK cell when part of a dimerized lymphoproliferative element
  • Intracellular domains from IFNAR1, IFNGR1, IFNLR1, IL2RB, IL4R, IL5RB, IL6R, IL6ST, IL7RA, IL9R, IL10RA, IL21R, IL27R, IL31RA, LIFR, and OSMR are known in the art to activate JAK1 signaling and thus comprise a JAK1 binding motif.
  • Intracellular domains from CRLF2, CSF2RA, CSF2RB, CSF3R, EPOR, GHR, IFNGR2, IL3RA, IL5RA, IL6ST, IL20RA, IL20RB, IL23R, IL27R, LEPR, MPL, and PRLR are known in the art to activate JAK2 and thus comprise a JAK2 binding motif.
  • Intracellular domains from IL2RG are known in the art to activate JAK3 and thus comprise a JAK3 binding motif.
  • Intracellular domains from GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IL2RB, IL2RG, IL4R, IL5RA, IL5RB, IL7RA, IL9R, IL21R, IL22RA1, IL31RA, LIFR, MPL, and OSMR are known in the art to activate STATE.
  • Intracellular domains from GHR, IL2RB, IL2RG, IL6R, IL7RA, IL9R, IL10RA, IL 1 ORB, IL21R, IL22RA1, IL23R, IL27R, IL31RA, LEPR, LIFR, MPL, and OSMR are known in the art to activate STAT3.
  • Intracellular domains from IL12RB1 are known in the art to activate STAT4.
  • Intracellular domains from CSF2RA, CSF2RB, CSF3R, EPOR, GHR, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL5RB, IL7RA, IL9R, IL15RA, IL20RA, IL20RB, IL21R, IL22RA1, IL31RA, LIFR, MPL, OSMR, and PRLR are known in the art to activate STAT5.
  • Intracellular domains from IL4R and OSMR are known in the art to activate STAT6.
  • genes and intracellular domains thereof that are found in a first intracellular domain are the same as the optional second intracellular domain, except that if the first and second intracellular domain are identical, then at least one, and typically both the transmembrane domain and the extracellular domain are not from the same gene.
  • a lymphoproliferative element herein can include one or more intracellular signaling domains that include one or more Boxl motifs.
  • the one or more intracellular signaling domains that include one or more Boxl motifs can be IL7RA (Boxl motif at residues 9-17 of SEQ ID NOs:248 and 249), IL12RB ((Boxl motifs at residues 10-12 of SEQ ID NOs:254 and 255; and residues 107-110 and 139-142 of SEQ ID NO:256), IL31RA (Boxl motifs at residues 12-15 of SEQ ID NOs:275 and 276), CSF2RB (Boxl motif at residues 14-22 of SEQ ID NO:213), IL2RB (Boxl motif at residues 13-21 of SEQ ID NO:240), IL6ST (Boxl motif at residues 10- 18 of SEQ ID NO:247), IL2RG (Boxl motif at residues 3-11 of SEQ ID NO:241),
  • a lymphoproliferative element herein can include one or more intracellular signaling domains that include one or more Box2 motifs.
  • the one or more intracellular signaling domains that include one or more Box2 motifs can be MPL (Box2 motif at residues 46-64 in SEQ ID NO:283), IFNAR2 (Boxl motif at residues 37-46 of SEQ ID NO:227), CSF3R, or EPOR (Box2 motif at residues 303-313 of full-length EPOR).
  • EPOR also contains an extended Box2 motif (residues 329-372 of full-length EPOR) important for binding tyrosine kinase receptor KIT, which, in some embodiments, a lymphoproliferative element can include.
  • CSF3R also contains a Box3 motif, which, in some embodiments, a lymphoproliferative element can include.
  • the Boxl motif-containing lymphoproliferative element has a switch motif, which in illustrative embodiments has one or more, and preferably all hydrophobic residues at positions -1, -2, and -6 relative to the Boxl motif.
  • the Boxl motif an ICD of a lymphoproliferative element is located proximal to the transmembrane (TM) domain (for example between 5 and 15 or about 10 residues downstream from the TM domain) relative to the Box2 motif, which is located proximal to the transmembrane domain (for example between 10 and 50 residues downstream from the TM domain) relative to the STAT binding motif.
  • the STAT binding motif typically comprising a tyrosine residue, the phosphorylation of which affects binding of a STAT to the STAT binding motif of the lymphoproliferative element.
  • the ICDs comprising multiple STAT binding motifs where multiple STAT binding motifs are present in a native ICD (e.g., EPO receptor and IL-6 receptor signaling chain (gpl30).
  • the switch motif containing intracellular signaling domain can be MPL (switch motif at residues 11, 15, and 16 of SEQ ID NO:283).
  • a lymphoproliferative element herein can include one or more intracellular signaling domains that include one or more phosphorylatable residues, for example, a phosphorylatable serine, threonine, or tyrosine.
  • the one or more intracellular signaling domains that include one or more phosphorylatable residues can be IL31RA (phosphorylatable tyrosines at residues Y96, Y237, and Y165 of SEQ ID NO:275; not present in SEQ ID NO:276), CD27 (phosphorylatable serine at residue S6 of SEQ ID NO:205), CSF2RB (phosphorylatable tyrosine at residue Y306 of SEQ ID NO:213), IL6ST (phosphorylatable serines at residues S20, S26, S141, S148, S188, and S198 of SEQ ID NO:247), MPL (phosphorylatable tyrosines at residues Y8, Y29, Y78, Y113, and Y118 of SEQ ID NO: 283), CD79B (phosphorylatable tyrosines at residues Y16 and Y27 of SEQ ID NO: 211), OSMR (phosphorylatable serines at residues Y16
  • a lymphoproliferative element that includes a CSF3R intracellular domain can include one, two, three, or all of the tyrosine residues corresponding to Y704, Y729, Y744, and Y764 of full-length CSF3R, various combinations of which have been shown to be important for binding Stat3, SOCS3, Grb2, and p21Ras.
  • a lymphoproliferative element herein can include one or more intracellular signaling domains that has one or more of its phosphorylatable residues mutated to a phosphomimetic residue, for example, aspartic acid or glutamic acid.
  • a lymphoproliferative element herein can include one or more intracellular signaling domains that has one or more of its phosphorylatable tyrosines mutated to a non-phosphorylatable residue, for example, alanine, valine, or phenylalanine.
  • a lymphoproliferative element that includes a CSF3R intracellular domain can include one or more mutations corresponding to T615A and T618I of full-length CSF3R, which have been shown to increase receptor dimerization and activity.
  • a lymphoproliferative element herein can include one or more intracellular signaling domains that include one or more ubiquitination targeting motif residues.
  • the one or more intracellular signaling domains that include one or more ubiquitination targeting motif residues can be MPL (residues at K40 and K60 of SEQ ID NO:283) or 0X40 (residues at K17 and K41 of SEQ ID NO:296).
  • an intracellular domain including ubiquitination targeting motif residues can have one or more of the lysines mutated to arginine or another amino acid.
  • a lymphoproliferative element herein can include one or more intracellular signaling domains that include one or more TRAF binding sites.
  • TRAF1, TRAF2, and TRAF3 binding sites include the amino acid sequence PXQXT (SEQ ID NO:303), where each X can be any amino acid, a distinct TRAF2 binding site includes the consensus sequence SXXE (SEQ ID NO:304) where each X can be any amino acid, and a TRAF6 binding site includes the consensus sequence QXPXEX (SEQ ID NO:305).
  • the one or more intracellular signaling domains that include one or more TRAF binding sites can be CD40 (binding sites for TRAF1, TRAF2, and TRAF3 at residues 35-39 of SEQ ID NO:208; TRAF2 binding site at residues 57-60 of SEQ ID NO:208; TRAF6 binding site at residues 16-21 of SEQ ID NO:208), or 0X40 (TRAF1, TRAF2, TRAF3, and TRAF5 binding motif at residues 20-27 of SEQ ID NO:296).
  • a lymphoproliferative element herein can include one or more intracellular signaling domains that include a TIR domain.
  • the one or more intracellular signaling domains that include a TIR domains can be IL17RE (TIR domain at residues 13-136 of SEQ ID NO:265), IL18R1 (TIR domain at residues 28-170 of SEQ ID NO:266), or MyD88 (TIR domain at residues 160-304 of SEQ ID NO:284).
  • a lymphoproliferative element herein can include one or more intracellular signaling domains that include a PI3K binding motif domain.
  • the one or more intracellular signaling domains that include a PI3K binding motif can be CD28 (PI3K binding motifs at residues 12-15 of SEQ ID NOs:206 and 207, which also binds Grb2), ICOS (PI3K binding motif at residues 19-22 of SEQ ID NO:225, which can be mutated F21Q to increase IL-2 production and/or to bind Grb2), 0X40 (p85 PI3K binding motif at residues 34-57 of full-length 0X40)
  • a lymphoproliferative element herein can include one or more intracellular signaling domains that include a dileucine motif.
  • the one or more intracellular signaling domains that include a dileucine motif can be IFNGR2 (dileucine motif at residues 8-9 of SEQ ID NO:230) or CD3G (dileucine motif at residues 131-132 of full-length CD3G).
  • one or both of the residues in the dileucine motif can be mutated.
  • a lymphoproliferative element herein can include one or more intracellular signaling domains that include one or more N-terminal death domains.
  • the one or more intracellular signaling domains that include one or more N-terminal death domains can be MyD88 (N-terminal death domain at residues 29-106 of SEQ ID NO:284) or a TNFR.
  • TNFRs TNF receptors
  • TNFRSF4 TNF receptors
  • TNFRSF8 TNF receptor-associated factors
  • DD death domain
  • the domains, motifs, and point mutations of TNFRs that induce proliferation and/or survival of T cells and/or NK cells are known in the art and a skilled artisan can identify corresponding domains, motifs, and point mutations in TNFR polypeptides.
  • a lymphoproliferative element that includes a TNFR intracellular domain can include one or more TRAF-binding motifs.
  • a lymphoproliferative element that includes a TNFR intracellular domain does not include a DD-binding motif, or has one or more DD-binding motifs deleted or mutated within the intracellular domain.
  • a lymphoproliferative element that includes a TNFR intracellular domain can recruit TRADD and/or TRAF2.
  • TNFRs also include cysteine -rich domains (CRDs) that are important for ligand binding (Locksley RM et al. Cell. 2001 Feb 23;104(4):487-501).
  • CRDs cysteine -rich domains
  • a lymphoproliferative element that includes a TNFR intracellular domain does not include a TNFR CRD.
  • a lymphoproliferative element herein can include one or more intracellular signaling domains that include one or more intermediate domains that interact with IL-1R associated kinase.
  • the one or more intracellular signaling domains that include one or more intermediate domains can be MyD88 (intermediate domain at residues 107-156 of SEQ ID NO:284), [0423]
  • a lymphoproliferative element that includes an intracellular domain from IL7RA can include one or more of the S region or T region (S region at residues 359-394 and T region at residues Y401, Y449, and Y456 of full-length IL7RA).
  • the second intracellular domain can be derived from TNFRSF8.
  • the second intracellular domain can be other than an intracellular domain derived from MyD88, a CD28 family member (e.g., CD28, ICOS), Pattern Recognition Receptor, a C- reactive protein receptor (i.e., Nodi, Nod2, PtX3-R), a TNF receptor, CD40, RANK/TRANCE-R, 0X40, 4-1BB), an HSP receptor (Eox-1 and CD91), or CD28.
  • a CD28 family member e.g., CD28, ICOS
  • Pattern Recognition Receptor e.e., a C- reactive protein receptor (i.e., Nodi, Nod2, PtX3-R), a TNF receptor, CD40, RANK/TRANCE-R, 0X40, 4-1BB), an HSP receptor (Eox-1 and CD91), or CD28.
  • Pattern Recognition Receptors include, but are not limited to endocytic pattern-recognition receptors (i.e., mannose receptors, scavenger receptors (i.e., Mac- 1, LRP, peptidoglycan, teichoic acids, toxins, CD1 1 c/CR4)); external signal pattern-recognition receptors (Toll-like receptors (TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10), peptidoglycan recognition protein, (PGRPs bind bacterial peptidoglycan, and CD 14); internal signal pattern-recognition receptors (i.e., NOD-receptors 1 & 2), and RIG1.
  • endocytic pattern-recognition receptors i.e., mannose receptors, scavenger receptors (i.e., Mac- 1, LRP, peptidoglycan, teichoic acids,
  • a lymphoproliferative element that includes an intracellular domain from MyD88 can include one or more of the mutations L93P, R193C, and L265P in full-length MyD88 (mutations L93P, R196C, and L260P of SEQ ID NO:284).
  • the second intracellular domain can be derived from TNFRSF4 or TNFRSF8.
  • the second intracellular domain can be other than an intracellular domain derived from a CD28 family member (e.g., CD28, ICOS), Pattern Recognition Receptor, a C-reactive protein receptor, a TNF receptor, or an HSP receptor.
  • a CD28 family member e.g., CD28, ICOS
  • Pattern Recognition Receptor e.g., a C-reactive protein receptor, a TNF receptor, or an HSP receptor.
  • a cell expressing the lymphoproliferative element comprising an intracellular and transmembrane domain of MPL can be contacted with or exposed to eltrombopag, or a patient or subject to which such a cell has been infused can be treated with eltrombopag.
  • eltrombopag binds to the transmembrane domain of MPL and induces the activation of the intracellular domain of MPL.
  • a MPL intracellular signaling domain does not comprise the region comprising amino acids 70-95 in SEQ ID NO:283.
  • the intracellular portion of MPL can include one or more, or all the domains and motifs described herein that are present in SEQ ID NO: 283.
  • a transmembrane portion of MPL can include one or more, or all the domains and motifs described herein that are present in SEQ ID NO: 187.
  • the second intracellular domain can be derived from CD79B.
  • the first intracellular domain can be derived from CSF3R.
  • a lymphoproliferative element that includes an PRLR intracellular domain can include the growth hormone receptor binding domain of PRLR and any known mutations (growth hormone receptor binding domain at residues 28-104 of SEQ ID NO:295).
  • a lymphoproliferative element that includes an ICOS intracellular domain can include a calcium-signaling motif (calcium-signaling motif at residues 5-8 of SEQ ID NO:225). In some embodiments, a lymphoproliferative element that includes an ICOS intracellular domain can include at least one of a first and second conserved motif (first and second conserved motifs at residues 9- 18 and 24-30, respectively, of SEQ ID NO:225). In some embodiments, a lymphoproliferative element that includes an ICOS intracellular domain does not include at least one of the first and second conserved motif.
  • EPOR also contains a short segment important for EPOR internalization (residues 267-276 of full-length EPOR).
  • a lymphoproliferative element that includes an EPOR intracellular domain does not include the internalization segment.
  • domains, motifs, and point mutations of intracellular signaling domains that induce proliferation and/or survival of T cells and/or NK cells are known in the art and a skilled artisan can identify corresponding domains, motifs, and point mutations in polypeptides, some of which are above, and a skilled artisan can identify corresponding domains, motifs, and point mutations in other polypeptides. A skilled artisan will be able to identify these domains, motifs, and point mutations in similar polypeptides using, for example, sequence alignments to known binding motifs.
  • a lymphoproliferative element herein can include any, for example, one or more up to all of the domains, motifs, and mutations of a intracellular signaling domain disclosed herein or otherwise known to induce proliferation and/or survival of T cells and/or NK cells.
  • the LE provides, is capable of providing and/or possesses the property of (or a cell modified, genetically modified, and/or transduced with the LE is capable of providing, is adapted for, possesses the property of, and/or is modified for) driving T cell expansion in vivo.
  • the lymphoproliferative element can include any of the sequences listed in Table 1 (SEQ ID NOs: 84-302). Table 1 shows the parts, names (including gene names), and amino acid sequences for domains that were tested in CLEs.
  • CLEs can include in certain illustrative embodiments, an extracellular domain (denoted Pl), a transmembrane domain (denoted P2), a first intracellular domain (denoted P3), and a second intracellular domain (denoted P4).
  • the lymphoproliferative element includes a first intracellular domain.
  • the first intracellular domain can include any of the parts listed as S036 to S0216 or in Table 1, or functional mutants and/or fragments thereof.
  • the lymphoproliferative element can include a second intracellular domain.
  • the second intracellular domain can include any of the parts listed as S036 to S0216 or in Table 1, or functional mutants and/or fragments thereof.
  • the lymphoproliferative element can include an extracellular domain.
  • the extracellular domain can include any of the sequences of parts listed as M001 to M049 or E006 to E015 in Table 1, or functional mutants and/or fragments thereof.
  • the lymphoproliferative element can include a transmembrane domain.
  • the transmembrane domain can include any of the parts listed as M001 to M049 or T001 to T082 in Table 1, or functional mutants and/or fragments thereof.
  • the lymphoproliferative element can be a fusion of an extracellular/transmembrane domain (M001 to M049 in Table 1), a first intracellular domain (S036 to S0216 in Table 1), and a second intracellular domain (S036 to S216 in Table 1).
  • the lymphoproliferative element can be a fusion of an extracellular domain (E006 to E016 in Table 1), a transmembrane domain (T001 to T082 in Table 1), a first intracellular domain (S036 to S0216 in Table 1), and a second intracellular domain (S036 to S0216 in Table 1).
  • the lymphoproliferative element can be a fusion of E006, T001, S036, and S216, also written as E006-T001-S036-S216).
  • the lymphoproliferative element can be the fusion E010-T072-S192-S212, E007-T054-S197-S212, E006-T006-S194-S211, E009-T073-S062-S053, E008-T001-S121-S212, E006- T044-S186-S053, or E006-T016-S186-S050.
  • the intracellular domain of an LE is other than a functional intracellular activating domain from an ITAM-containing intracellular domain, for example, an intracellular domain from CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP12, FCER1G, FCGR2A, FCGR2C, DAP10/CD28, or ZAP70, and in a further illustrative subembodiment, CD3z.
  • the extracellular domain of an LE does not comprise a single-chain variable fragment (scFv).
  • the extracellular domain of an LE that upon binding to a binding partner activates an LE does not comprise a single -chain variable fragment (scFv).
  • a CLE does not comprise both an ASTR and an activation domain from CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP12, FCER1G, FCGR2A, FCGR2C, DAP10/CD28, or ZAP70. If an LE does include an ASTR (and not an activation domain in the previous list), the ASTR of an LE in illustrative embodiments does not include an scFv. In some embodiments, a lymphoproliferative element does not include an extracellular domain.
  • the lymphoproliferative element, and in illustrative embodiments CLE is not covalently attached to a cytokine.
  • a lymphoproliferative element, and in illustrative embodiments CLE comprises a cytokine polypeptide covalently linked to its cognate receptor.
  • the CLE can be constitutively active and typically constitutively activates the same Jak/STAT and/or TRAF pathways as the corresponding activated wild-type cytokine receptor.
  • the chimeric cytokine receptor is an interleukin.
  • the CLE is IL-7 covalently linked to IL7RA or IL-15 covalently linked to IL15RA. In other embodiments, the CLE is other than IL-15 covalently linked to IL15RA. In other aspects, the CLE comprises a cytokine polypeptide covalently linked to only a portion of its cognate receptor that includes a functional portion of the extracellular domain capable of binding the cytokine polypeptide, the transmembrane domain and/or intracellular domain are from heterologous polypeptides, and the CLE is constitutively active. In one embodiment, the CLE is IL-7 covalently linked to the extracellular and transmembrane domains of IL7RA, and the intracellular domain from IL2RB.
  • the CLE is a cytokine polypeptide covalently linked to a portion of its cognate receptor that includes a functional portion of the extracellular domain capable of binding the cytokine polypeptide, a heterologous transmembrane domain, and lymphoproliferative element intracellular domain provided herein.
  • the lymphoproliferative element is a cytokine receptor that is not tethered to a cytokine.
  • the lymphoproliferative element is capable of binding to soluble cytokines or growth factors and such binding is required for activity.
  • the lymphoproliferative element is constitutively active, and thus does not require binding to a soluble growth factor or cytokine for activity.
  • constitutively active lymphoproliferative elements do not bind soluble cytokines or growth factors.
  • the lymphoproliferative element is a chimera comprising an extracellular binding domain from one receptor and the intracellular signaling domain from a different receptor.
  • the CLE is an inverted receptor that is activated upon binding of a ligand that would inhibit proliferation and/or survival when bound to its natural receptor, but instead leads to proliferation and/or survival upon activating the CLE.
  • inverted receptors include chimeras that comprise an extracellular ligand binding domain from IL4Ra and an intracellular domain from IL7Ra or IL21.
  • Other embodiments of inverted cytokine receptors include chimeras that comprise an extracellular ligand binding domain from a receptor that would inhibit proliferation and/or survival when bound to its natural ligand, such as receptors for IL-4, IL-10, IL-13, or TGFb, and any lymphoproliferative element intracellular domain disclosed herein.
  • the lymphoproliferative element does not bind a cytokine. In further illustrative aspects, the lymphoproliferative element does not bind any ligand. In illustrative embodiments, the lymphoproliferative elements that do not bind any ligand are constitutively dimerized or otherwise multimerized, and are constitutively active. In illustrative embodiments of any of the methods and compositions provided herein that include a lymphoproliferative element, the intracellular domain can be derived from an intracellular portion of the transmembrane protein of the TNF receptor family, CD40.
  • the domains, motifs, and point mutations of CD40 that induce proliferation and/or survival of T cells and/or NK cells are known in the art and a skilled artisan can identify corresponding domains, motifs, and point mutations in CD40 polypeptides, some of which are discussed in this paragraph.
  • the CD40 protein contains several binding sites for TRAF proteins. Not to be limited by theory, binding sites for TRAF1, TRAF2, and TRAF3 are located at the membrane distal domain of the intracellular portion of CD40 and include the amino acid sequence PXQXT (SEQ ID NO:303) where each X can be any amino acid, (corresponding to amino acids 35-39 of SEQ ID NO:208) (Elgueta et al. Immunol Rev. 2009 May;
  • TRAF2 has also been shown to bind to the consensus sequence SXXE (SEQ ID NO:304) where each X can be any amino acid, (corresponding to amino acids 57-60 of SEQ ID NO:208) (Elgueta et al. Immunol Rev. 2009 May; 229(1): 152-72).
  • SXXE consensus sequence
  • a distinct binding site for TRAF6 is situated at the membrane proximal domain of intracellular portion of CD40 and includes the consensus sequence QXPXEX (SEQ ID NO:305) where each X can be any amino acid (corresponding to amino acids 16-21 of SEQ ID NO:208) (Lu et al. J Biol Chem. 2003 Nov 14; 278(46):45414-8).
  • the intracellular portion of the transmembrane protein CD40 can include all the binding sites for the TRAF proteins.
  • the TRAF binding sites are known in the art and a skilled artisan will be able to identify corresponding TRAF binding sites in similar CD40 polypeptides.
  • a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:208 or SEQ ID NO:209.
  • the intracellular domain derived from CD40 has a length of from about 30 amino acids (aa) to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, or from about 60 aa to about 65 aa.
  • the intracellular domain derived from CD40 has a length of from about 30 aa to about 66 aa, for example, 30 aa to 65 aa, or 50 aa to 66 aa.
  • the second intracellular domain can be other than an intracellular domain derived from MyD88, a CD28 family member (e.g., CD28, ICOS), Pattern Recognition Receptor, a C-reactive protein receptor (i.e., Nodi, Nod2, PtX3-R), a TNF receptor, CD40, RANK/TRANCE-R, 0X40, 4-1BB), an HSP receptor (Lox-1 and CD91), or CD28.
  • a CD28 family member e.g., CD28, ICOS
  • Pattern Recognition Receptor e., a C-reactive protein receptor (i.e., Nodi, Nod2, PtX3-R), a TNF receptor, CD40, RANK/TRANCE-R, 0X40, 4-1BB), an HSP receptor (Lox-1 and CD91), or CD28.
  • Pattern Recognition Receptors include, but are not limited to endocytic pattern-recognition receptors (i.e., mannose receptors, scavenger receptors (i.e., Mac-1, LRP, peptidoglycan, teichoic acids, toxins, CD1 1 c/CR4)); external signal pattern-recognition receptors (Toll-like receptors (TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10), peptidoglycan recognition protein, (PGRPs bind bacterial peptidoglycan, and CD14); internal signal pattern-recognition receptors (i.e., NOD-receptors 1 & 2), and RIG1.
  • endocytic pattern-recognition receptors i.e., mannose receptors, scavenger receptors (i.e., Mac-1, LRP, peptidoglycan, teichoic acids,
  • the intracellular domain can be derived from a portion of the transmembrane protein MPL.
  • the lymphoproliferative element comprises MPL, or is MPL, or a variant and/or fragment thereof, including a variant and/or fragment that includes at least 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% of the intracellular domain of MPL, with or without a transmembrane and/or extracellular domain of MPL, wherein the variant and/or fragment retains the ability to promote cell proliferation of PBMCs, and in some embodiments T cells.
  • a cell expressing the lymphoproliferative element comprising an intracellular and transmembrane domain of MPL can be contacted with, exposed to, or treated with eltrombopag.
  • eltrombopag binds to the transmembrane domain of MPL and induces the activation of the intracellular domain of MPL.
  • the domains, motifs, and point mutations of MPL that induce proliferation and/or survival of T cells and/or NK cells are known in the art and a skilled artisan can identify corresponding domains, motifs, and point mutations in MPL polypeptides, some of which are discussed in this paragraph.
  • the transmembrane MPL protein contains the Boxl motif PXXP (SEQ ID NO:306) where each X can be any amino acid (corresponding to amino acids 17-20 in SEQ ID NO:283) and the Box2 motif, a region with increased serine and glutamic acid content (corresponding to amino acids 46-64 in SEQ ID NO:283) (Drachman and Kaushansky. Proc Natl Acad Sci U S A. 1997 Mar 18; 94(6):2350-5).
  • the Boxl and Box2 motifs are involved in binding to JAKs and signal transduction, although the Box2 motif presence is not always required for a proliferative signal (Murakami et al. Proc Natl Acad Sci U S A.
  • cytokine receptors have hydrophobic residues at positions -1, -2, and -6 relative to the Boxl motif (corresponding to amino acids 16, 15, and 11, respectively, of SEQ ID NO:283), that form a “switch motif,” which is required for cytokine-induced JAK2 activation but not for JAK2 binding (Constantinescu et al. Mol Cell. 2001 Feb; 7(2):377-85; and Huang et al. Mol Cell.
  • a MPL intracellular signaling domain does not comprise the region comprising amino acids 70-95 in SEQ ID NO:283.
  • the lysines K553 corresponding to K40 of SEQ ID NO: 283
  • K573 corresponding to K60 of SEQ ID NO: 283
  • a MPL intracellular signaling domain does not comprise these ubiquitination targeting motif residues.
  • the tyrosines Y521 (corresponding to Y8 of SEQ ID NO: 283), Y542 (corresponding to Y29 of SEQ ID NO:283), Y591 (corresponding to Y78 of SEQ ID NO: 283), Y626 (corresponding to Y113 of SEQ ID NO: 283), and Y631 (corresponding to Y118 of SEQ ID NO: 283) have been shown to be phosphorylated (Varghese et al. Front Endocrinol (Lausanne). 2017 Mar 31; 8:59).
  • Y521 and Y591 of full-length MPL are negative regulatory sites that function either as part of a lysosomal targeting motif (Y521) or via an interaction with adaptor protein AP2 (Y591) (Drachman and Kaushansky. Proc Natl Acad Sci U S A. 1997 Mar 18; 94(6):2350-5; and Hitchcock et al. Blood. 2008 Sep 15; 112(6):2222-31).
  • Y626 and Y631 of full-length MPL are positive regulatory sites (Drachman and Kaushansky. Proc Natl Acad Sci U S A.
  • MPL contains the She phosphotyrosine-binding binding motif NXXY (SEQ ID NO:307) where each X can be any amino acid (corresponding to amino acids 110-113 of SEQ ID NO: 283), and this tyrosine is phosphorylated and important for the TPO- dependent phosphorylation of She, SHIP, and STAT3 (Laminet et al. J Biol Chem. 1996 Jan 5; 271(l):264-9; and van der Geer et al. Proc Natl Acad Sci U S A. 1996 Feb 6; 93(3):963-8).
  • MPL also contains the STAT3 consensus binding sequence YXXQ (SEQ ID NO:308) where each X can be any amino acid (corresponding to amino acids 118-121 of SEQ ID NO: 283) (Stahl et al. Science. 1995 Mar 3; 267(5202): 1349-53).
  • the tyrosine of this sequence can be phosphorylated and MPL is capable of partial STAT3 recruitment (Drachman and Kaushansky. Proc Natl Acad Sci U S A. 1997 Mar 18; 94(6):2350-5).
  • MPL also contains the sequence YLPL (SEQ ID NO: 309) (corresponding to amino acid 113-116 of SEQ ID NO: 283), which is similar to the consensus binding site for STAT5 recruitment pYLXL (SEQ ID NOG 10) where pY is phosphotyrosine and X can be any amino acid (March et al. FEBS Lett. 1996 Sep 30; 394(2):221 -6). Using computer simulations, Lee et al.
  • the intracellular portion of MPL can include one or more, or all the domains and motifs described herein that are present in SEQ ID NO 283.
  • a transmembrane portion of MPL can include one or more, or all the domains and motifs described herein that are present in SEQ ID NO: 187.
  • the domains, motifs, and point mutations of MPL provided herein are known in the art and a skilled artisan would recognize that MPL intracellular signaling domains herein in illustrative embodiments would include one or more corresponding domains, motifs, and point mutations in that have been shown to promote proliferative activity and would not include that that have been shown to inhibit MPLs proliferative activity.
  • a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:283.
  • the intracellular domain derived from MPL has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 100 aa, from about 100 aa to about 125 aa, from about 125 aa to 150 aa, from about 150 to about 175 aa, from about 175 aa to about 200 aa, from about 200 aa to about 250 aa, from about 250 aa to 300 aa, from about 300 aa to 350 aa, from about 350 aa to about 400 aa, from about 400 aa to about 450 aa, from about
  • the intracellular domain derived from MPL has a length of from about 30 aa to about 200 aa, for example, 30 aa to 150 aa, 30 aa to 119 aa, 30 aa to 121 aa, 30 aa to 122 aa, or 50 aa to 125 aa.
  • the second intracellular domain can be derived from CD79B.
  • Lymphoproliferative elements and CLEs that can be included in any of the aspects disclosed herein, can be any of the LEs or CLEs disclosed in WO2019/055946.
  • CLEs were disclosed therein that promoted proliferation in cell culture of PBMCs that were transduced with lentiviral particles encoding the CLEs between day 7 and day 21, 28, 35 and/or 42 after transduction.
  • CLEs were identified therein, that promoted proliferation in vivo in mice in the presence or absence of an antigen recognized by a CAR, wherein T cells expressing one of the CLEs and the CAR were introduced into the mice.
  • tests and/or criteria can be used to identify whether any test polypeptide, including LEs, or test domains of an LE, such as a first intracellular domain, or a second intracellular domain, or both a first and second intracellular domain, are indeed LEs or effective intracellular domains of LEs, or especially effective LEs or intracellular domains of LEs.
  • any aspect or other embodiment provided herein that includes an LE or a polynucleotide or nucleic acid encoding an LE can recite that the LE meets, or provides the property of, or is capable of providing and/or possesses the property of, any one or more of the identified tests or criteria for identifying an LE provided herein, or that a cell genetically modified, transduced, and/or stably transfected with a recombinant nucleic acid vector, such as a cell that is transduced with a lentiviral particle encoding the LE, is capable of providing, is adapted for, possesses the property of, and/or is modified for achieving the results of one or more of the recited tests.
  • the LE provides, is capable of providing and/or possesses the property of, (or a cell genetically modified and/or transduced with a retroviral particle encoding the LE is capable of providing, is adapted for, possesses the property of, and/or is modified for) improved expansion to pre-activated PBMCs transduced with a lentivirus comprising a nucleic acid encoding the LE and an anti-CD19 CAR comprising a CD3 zeta intracellular activating domain but no co-stimulatory domain, between day 7 and day 21, 28, 35, and/or 42 of in vitro culturing post-transduction in the absence of exogenously added cytokines, compared to a control retroviral particle, e.g.
  • a lymphoproliferative element test for improved or enhanced survival, expansion, and/or proliferation of cells transduced with a retroviral particle e.g. lentiviral particle
  • a retroviral particle e.g. lentiviral particle
  • test cells can be performed based on a comparison to control cells, which can be, for example, either untransduced cells or cells transduced with a control retroviral (e.g.
  • control cells are transduced with a retroviral particle (e.g., lentiviral particle) having a genome encoding a lymphoproliferative element or intracellular domain(s) thereof, identified herein as exemplifying a lymphoproliferative element.
  • a retroviral particle e.g., lentiviral particle
  • the test criteria can include that there is at least as much enrichment, survival and/or expansion, or no statistical difference of enrichment, survival, and/or expansion when the test is performed using a retroviral particle (e.g., lentiviral particle) having a genome encoding a test construct versus encoding the control lymphoproliferative element, typically by analyzing cells transcribed therewith.
  • a retroviral particle e.g., lentiviral particle
  • Exemplary or illustrative embodiments of lymphoproliferative elements herein, in some embodiments, are illustrative embodiments of control lymphoproliferative elements for such a test.
  • this test for an improved property of a putative or test lymphoproliferative element is performed by performing replicates and/or performing a statistical test.
  • a skilled artisan will recognize that many statistical tests can be used for such a lymphoproliferative element test. Contemplated for such a test in these embodiments would be any such test known in the art.
  • the statistical test can be a T-test or a Mann-Whitney-Wilcoxon test.
  • the normalized enrichment level of a test construct is significant at a p-value of less than 0.1 , or less than 0.05, or less than 0.01.
  • the LE provides, is capable of providing and/or possesses the property of (or a cell genetically modified and/or transduced with the LE is capable of providing, is adapted for, possesses the property of, and/or is modified for) at least a 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold expansion, or between 1.5 fold and 25-fold expansion, or between 2-fold and 20-fold expansion, or between 2-fold and 15-fold expansion, or between 5-fold and 25-fold expansion, or between 5-fold and 20-fold expansion, or between 5-fold and 15-fold expansion, of preactivated PBMCs transduced with a nucleic acid encoding the LE when transduced along with an antiCD 19 CAR comprising a CD3 zeta intracellular activating domain but no co-stimulatory domain, between day 7 and day 21, 28, 35, and/or 42 of in vitro culturing in the absence of exogenously added cytok
  • the test is performed in the presence of PBMCs, for example at a 1 : 1 ratio of transduced cells to PBMCs, which can be for example, from a matched donor, and in some embodiments, the test is performed in the absence of PBMCs.
  • the analysis of expansion for any of these tests is performed as illustrated in WO2019/055946.
  • the test can include a further statistical test and a cut-off such as a P value below 0.1, 0.05, or 0.01, wherein a test polypeptide or nucleic acid encoding the same, needs to meet one or both thresholds (i.e., fold expansion and statistical cutoff).
  • the number of test cells and the number of control cells can be compared between day 7 and day 14, 21, 28, 35, 42 or 60 posttransduction.
  • the numbers of test and control cells can be determined by sequencing DNA and counting the occurrences of unique identifiers present in each construct.
  • the numbers of test and control cells can be counted directly, for example with a hemocytometer or a cell counter.
  • all the test cells and control cells can be grown within the same vessel, well or flask.
  • test cells can be seeded in one or more wells, flasks or vessels, and the control cells can be seeded in one or more flasks or vessels.
  • test and control cells can be seeded individually into wells or flasks, e.g., one cell per well.
  • the numbers of test cells and control cells can be compared using enrichment levels.
  • the enrichment level for a test or control construct can be calculated by dividing the number of cells at a later time point (day 14, 21, 28, 35, or day 45) by the number of cells at day 7 for each construct.
  • the enrichment level for a test or control construct can be calculated by dividing the number of cells at a time point (day 14, 21, 28, 35, or day 45) by the number of cells at that time point for untransduced cells.
  • the enrichment level of each test construct can be normalized to the enrichment level of the respective control construct to generate a normalized enrichment level.
  • a LE encoded in the test construct provides (or a cell genetically modified and/or transduced with a retroviral particle (e.g.
  • lentiviral particle) having a genome encoding the LE is capable of providing, is adapted for, possesses the property of, and/or is modified for) at least a 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold normalized enrichment level, or between 1.5 fold and 25-fold normalized enrichment level, or between 3-fold and 20- fold normalized enrichment level, or between 5-fold and 25-fold normalized enrichment level, or between 5-fold and 20-fold normalized enrichment level, or between 5-fold and 15-fold normalized enrichment level.
  • Enrichment can be measured, for example, by direct cell counting.
  • Cutoff values can be based on a single test, or two, three, four, or five repeats, or based on many repeats.
  • the cutoff can be met when a lymphoproliferative element meets one or more repeat tests, or meets or exceeds a cutoff for all repeats.
  • the enrichment is measured as log2((normalized count data on the test day + l)/(normalized count data on day 7 + 1)).
  • test constructs were identified as CLEs because the CLEs induced proliferation/expansion in these fed or unfed cultures without added cytokines such as IL-2 between days 7 and day 21, 28, 35, and/or 42.
  • effective CLEs were identified by identifying test CLEs that provided increased expansion of these in vitro cultures, whether fed or unfed with untransduced PBMCs, between day 7 and day 21, 28, 35, and/or 42 post-transduction, compared to control constructs that did not include any intracellular domains.
  • WO2019/055946 discloses that at least one and typically more than one test CLE that included an intracellular domain from a test gene provided more expansion than every control construct that was present at day 7 post-transduction, that did not include an intracellular domain.
  • WO2019/055946 further provides a statistical method that was used to identify exceptionally effective genes with respect to a first intracellular domain, and one or more exemplary intracellular domain(s) from these genes. The method used a Mann-Whitney-Wilcoxon test and a false discovery cutoff rate of less than 0.1 or less than 0.05.
  • WO2019/055946 identified especially effective genes for the first intracellular domain or the second intracellular domain, for example, by analyzing scores for genes calculated as combined score for all constructs with that gene. Such analysis can use a cutoff of greater than 1 , or greater than negative control constructs without any intracellular domains, or greater than 2, as shown for some of the tests disclosed in WO2019/055946.
  • the LE provides, is capable of providing and/or possesses the property of (or a cell genetically modified and/or transduced with the LE is capable of providing, is adapted for, possesses the property of, and/or is modified for) driving T cell expansion in vivo.
  • the in vivo test can utilize a mouse model and measure T cell expansion at 15 to 25 days in vivo, or at 19 to 21 days in vivo, or at approximately 21 days in vivo, after T cells are contacted with lentiviral vectors encoding the LEs, are introduced into the mice, as disclosed in WO2019/055946, [0446]
  • a LE which typically include a CAR, such as methods provided herein for modifying, genetically modifying and/or transducing cells, and uses thereof
  • the genetically modified cell is modified so as to possess new properties not previously possessed by the cell before genetic modification and/or transduction.
  • Such a property can be provided by genetic modification with a nucleic acid encoding a CAR or a LE, and in illustrative embodiments both a CAR and a LE.
  • the genetically modified and/or transduced cell is capable of, is adapted for, possesses the property of, and/or is modified for survival and/or proliferation in ex vivo culture for at least 7, 14, 21, 28, 35, 42, or 60 days or from between day 7 and day 14, 21, 28, 35, 42 or 60 post-transduction, in the absence of added IL-2 or in the absence of added cytokines such as IL-2, IL-15, or IL-7, and in certain illustrative embodiments, in the presence of the antigen recognized by the CAR where the method comprises modifying using a retroviral particle having a pseudotyping element and optionally a separate or fused activation domain on its surface and typically does not require preactivation.
  • the genetically modified and/or transduced cell exhibits, is capable of, is adapted for, possesses the property of, and/or is modified for improved survival or expansion in ex vivo or in vitro culture in culture media in the absence of one or more added cytokines such as IL-2, IL-15, or IL-7, or added lymphocyte mitogenic agent, compared to a control cell(s) identical to the genetically modified and/or transduced cell(s) before it was genetically modified and/or transduced or to a control cell that was transduced with a retroviral particle identical to an on-test retroviral particle that comprises an LE or a putative LE, but without the LE or the intracellular domains of the LE, wherein said survival or proliferation of said control cell(s) is promoted by adding said one or more cytokines, such as IL-2, IL-15, or IL-7, or said lymphocyte mitogenic agent to the culture media.
  • cytokines such as IL-2, IL-15, or IL-7
  • cytokine or lymphocyte mitogenic agent By added cytokine or lymphocyte mitogenic agent, it is meant that cytokine or lymphocyte mitogenic agent is added from an exogenous source to a culture media such that the concentration of said cytokine or lymphocyte mitogenic agent is increased in the culture media during culturing of the cell(s) compared to the initial culture media, and in some embodiments can be absent from the initial culture media before said adding.
  • added or exogenously added it is meant that such cytokine or lymphocyte mitogenic agent is added to a lymphocyte media used to culture the modified, genetically modified, and/or transduced cell after the modifying, where the culture media may or may not already possess the cytokine or lymphocyte mitogenic agent.
  • All or a portion of the media that includes a mixture of multiple media components is typically stored and in illustrative embodiments has been shipped to a site where the culturing takes place, without the exogenously added cytokine(s) or lymphocyte mitogenic agent(s).
  • the lymphocyte media in some embodiments is purchased from a supplier, and a user such as a technician not employed by the supplier and not located within a supplier facility, adds the exogenously added cytokine or lymphocyte mitogenic agent to the lymphocyte media and then the genetically modified and/or transduced cells are cultured in the presence or absence of such exogenously added cytokine or lymphocyte mitogenic agent.
  • improved or enhanced survival, expansion, and/or proliferation can be shown as an increase in the number of cells determined by sequencing DNA from cells transduced with retroviral particle (e.g. lentiviral particle) having a genome encoding CLEs and counting the occurrences of sequences present in unique identifiers from each CLE.
  • retroviral particle e.g. lentiviral particle
  • improved survival and/or improved expansion can be determined by counting the cells directly, for example with a hemocytometer or a cell counter, at each time point.
  • improved survival and/or improved expansion and/or enrichment can be calculated by dividing the number of cells at the later time point (day 21, 28, 35, and/or day 45) by the number of cells at day 7 for each construct.
  • the cells can be counted by hemocytometer or cell counters.
  • the enrichment level determined using the nucleic acid counts or the cell counts of each specific test construct can be normalized to the enrichment level of the respective control construct, i.e., the construct with the same extracellular domain and transmembrane domain but lacking the intracellular domains present in the test construct.
  • the LE encoded in the construct provides (or a cell genetically modified and/or transduced with a retroviral particle (e.g.
  • lentiviral particle) having a genome encoding the LE is capable of providing, is adapted for, possesses the property of, and/or is modified for) at least a 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold normalized enrichment level, or between 1.5 fold and 25-fold normalized enrichment level, or between 3-fold and 20-fold normalized enrichment level, or between 5-fold and 25-fold normalized enrichment level, or between 5- fold and 20-fold normalized enrichment level, or between 5-fold and 15-fold normalized enrichment level.
  • the lymphoproliferative element can include a cytokine receptor or a fragment that includes a signaling domain thereof.
  • the cytokine receptor can be CD27, CD40, CRLF2, CSF2RA, CSF2RB, CSF3R, EPOR, GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2R, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R, IL6ST, IL7R, IL7RA, IL9R, IL10RA, IL10RB, IL11RA, IL12RB1, IL13R, IL13RA1, IL13RA2, IL15R, IL15RA, IL17RA, IL17RB, IL17RC, IL17RE, IL18R1,
  • a lymphoproliferative element comprises an intracellular activating domain as disclosed hereinabove.
  • a lymphoproliferative element is a CLE comprising an intracellular activating domain comprising an ITAM-containing domain, as such, the CLE can comprise an intracellular activating domain having at least 80%, 90%, 95%, 98%, or 100% sequence identity to the CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP12, FCER1G, FCGR2A, FCGR2C, DAP10/CD28, or ZAP70 domains provided herein wherein the CLE does not comprise an ASTR.
  • one or more domains of a lymphoproliferative element is fused to a modulatory domain, such as a co-stimulatory domain, and/or an intracellular activating domain of a CAR.
  • a modulatory domain such as a co-stimulatory domain
  • an intracellular activating domain of a CAR can be part of the same polypeptide as a CAR or can be fused and optionally functionally connected to some components of CARs.
  • an engineered signaling polypeptide can include an ASTR, an intracellular activation domain (such as a CD3 zeta signaling domain), a co-stimulatory domain, and a lymphoproliferative domain. Further details regarding co-stimulatory domains, intracellular activating domains, ASTRs and other CAR domains, are disclosed elsewhere herein.
  • Lymphoproliferative elements typically include a transmembrane domain.
  • the transmembrane domain can have 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to any one of the transmembrane domains from the following genes and representative sequences disclosed in WO2019/055946: CD8 beta, CD4, CD3 zeta, CD28, CD134, CD7, CD2, CD3D, CD3E, CD3G, CD3Z, CD4, CD8A CD8B, CD27, CD28, CD40, CD79A, CD79B, CRLF2, CRLF2, CSF2RA, CSF2RB, CSF2RB, CSF3R, EPOR, FCER1G, FCGR2C, FCGRA2, GHR, ICOS, IFNAR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2,
  • Transmembrane (TM) domains suitable for use in any engineered signaling polypeptide include, but are not limited to, constitutively active cytokine receptors, the TM domain from LMP1, and TM domains from type 1 TM proteins comprising a dimerizing motif, as discussed in more detail herein.
  • the transmembrane domain can be a Type I growth factor receptor, a hormone receptor, a T cell receptor, or a TNF-family receptor.
  • CLEs include both an extracellular portion and a transmembrane portion that is from the same protein, in illustrative embodiments the same receptor, either of which in illustrative embodiments is a mutant, thus forming an extracellular and transmembrane domain.
  • These domains can be from a cytokine receptor, or a mutant thereof, or a hormone receptor, or a mutant thereof in some embodiments that have been reported to be constitutively active when expressed at least in some cell types.
  • such extracellular and transmembrane domains do not include a ligand binding region.
  • a mutation in at least some extracellular - transmembrane domains of CLEs provided herein are responsible for signaling of the CLE in the absence of ligand, by bringing activating chains together that are not normally together, or by changing the confirmation of a linked transmembrane and/or intracellular domain.
  • Exemplary extracellular and transmembrane domains for CLEs of embodiments that include such domains are extracellular regions, typically less than 30 amino acids of the membrane -proximal extracellular domains along with transmembrane domains from mutant receptors that have been reported to be constitutive, that is not require ligand binding for activation of an associated intracellular domain.

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Abstract

La présente divulgation concerne des procédés et des compositions qui comprennent un polynucléotide qui comprend des acides nucléiques qui codent un polypeptide anti-idiotype, ainsi que des polypeptides qui sont codés par ceux-ci, et des cellules qui comprennent et expriment le polypeptide. Les procédés divulgués comprennent des procédés qui utilisent les polypeptides anti-idiotypes en tant que commutateurs de sécurité lorsqu'ils sont utilisés en combinaison avec des anticorps, notamment des anticorps biologiques autorisés, qui comprennent l'idiotype reconnu. Certains modes de réalisation comprennent des polypeptides anti-idiotypes et des nucléotides les codant, qui comprennent un domaine interne. Dans certains modes de réalisation, ce domaine interne comprend des domaines fonctionnels qui peuvent induire la prolifération ou la mort cellulaire lors de la liaison du polypeptide anti-idiotype par son anticorps cible.
PCT/US2021/048532 2016-03-19 2021-08-31 Compositions anti-idiotype et procédés d'utilisation associés Ceased WO2022047417A1 (fr)

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CN202180052913.6A CN116249559A (zh) 2020-08-31 2021-08-31 抗独特型组合物及其使用方法
EP21862987.1A EP4204004A4 (fr) 2020-08-31 2021-08-31 Compositions anti-idiotype et procédés d'utilisation associés
US18/043,465 US20230357436A1 (en) 2016-03-19 2021-08-31 Anti-idiotype compositions and methods of use thereof
PCT/US2022/018404 WO2022187289A1 (fr) 2021-03-01 2022-03-01 Procédés et compositions pour l'administration de particules rétrovirales
CA3212366A CA3212366A1 (fr) 2021-03-01 2022-03-01 Procedes et compositions pour l'administration de particules retrovirales
AU2022229358A AU2022229358A1 (en) 2021-03-01 2022-03-01 Methods and compositions for the delivery of retroviral particles
US17/684,405 US20230044451A1 (en) 2016-03-19 2022-03-01 Methods and compositions for the delivery of modified lymphocytes and/or retroviral particles
EP22715804.5A EP4301862A1 (fr) 2021-03-01 2022-03-01 Procédés et compositions pour l'administration de particules rétrovirales
MX2023010059A MX2023010059A (es) 2021-03-01 2022-03-01 Metodos y composiciones para la administracion de particulas retrovirales.
JP2023553055A JP2024510933A (ja) 2021-03-01 2022-03-01 レトロウイルス粒子の送達のための方法および組成物

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WO2023196947A3 (fr) * 2022-04-07 2023-11-09 Board Of Regents, The University Of Texas System Procédés d'activation et d'expansion de cellules tueuses naturelles modifiées et combinaisons avec des anticorps
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WO2023196947A3 (fr) * 2022-04-07 2023-11-09 Board Of Regents, The University Of Texas System Procédés d'activation et d'expansion de cellules tueuses naturelles modifiées et combinaisons avec des anticorps

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