WO2024008177A1 - Cellules modifiées et leurs utilisations - Google Patents

Cellules modifiées et leurs utilisations Download PDF

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Publication number
WO2024008177A1
WO2024008177A1 PCT/CN2023/106274 CN2023106274W WO2024008177A1 WO 2024008177 A1 WO2024008177 A1 WO 2024008177A1 CN 2023106274 W CN2023106274 W CN 2023106274W WO 2024008177 A1 WO2024008177 A1 WO 2024008177A1
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Prior art keywords
binding moiety
amino acid
cell
acid sequence
seq
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PCT/CN2023/106274
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English (en)
Inventor
Li Ma
Ming Zeng
Cuiying SHAO
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Nanjing Curegene Technology Co., Ltd.
Nanjing Legend Biotech Co., Ltd.
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Publication of WO2024008177A1 publication Critical patent/WO2024008177A1/fr

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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
    • C07K16/2866—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 cytokines, lymphokines, interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to engineered cells comprising a surface molecule useful for treating infectious diseases such as HIV.
  • CCR5 The chemokine (C-C motif) receptor 5 (CCR5) serves as an HIV-1 co-receptor and is essential for cell infection with CCR5-tropic viruses. Loss of functional receptor protects against HIV infection. See Gupta et al. Nature volume 568, pages 244–248 (2019) . CCR5 gene-edited hematopoietic stem cells and progenitor cell (HSPC) transplantation is a promising strategy for HIV remission. However, only a fraction of HSPCs can be edited ex vivo to provide protection against infection prior to autologous transplantation.
  • HSPC progenitor cell
  • the present application in one aspect provides a surface molecule comprising:
  • a membrane domain that can tether the surface molecule to a cell membrane or facilitate the tethering of the surface molecule to cell membrane after being expressed in a cell, wherein the membrane domain comprises a Glycosylphosphatidylinositol (GPI) attachment signal sequence.
  • GPI Glycosylphosphatidylinositol
  • T cell surface antigen is CCR5.
  • the anti-HIV antibody is 10E8.
  • the first binding moiety specifically binds to CCR5 competitively with C1-13 or C1-14, or specifically binds to the same epitope as that of C1-13 or C1-14.
  • the first binding moiety comprises an antibody moiety comprising a heavy chain variable region (V H ) and a light chain variable region (V L )
  • V H comprises a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 3, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 5
  • the V L comprises a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8.
  • the V H in the first binding moiety is fused to the N-terminus of the V L . In other embodiments, in the first binding moiety the V H is fused to the C-terminus of the V L .
  • the V H of the first binding moiety comprises the amino acid sequence of SEQ ID NO: 32 or a variant comprising an amino acid sequence having at least about 80%sequence identity
  • the V L of the first binding moiety comprises the amino acid sequence of SEQ ID NO: 33 or a variant comprising an amino acid sequence having at least about 80%sequence identity.
  • the antibody moiety of the first binding moiety comprises an amino acid sequence of SEQ ID NO: 1 or 2, or a variant comprising an amino acid sequence having at least about 80%sequence identity.
  • the second binding moiety comprises an antibody moiety comprising a heavy chain variable region (V H ) and a light chain variable region (V L )
  • V H comprises a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 28
  • V L comprises a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 29, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 31.
  • the V H in the second binding moiety is fused to the N-terminus of the V L . In other embodiments, in the second binding moiety the V H is fused to the C-terminus of the V L .
  • the V H of the second binding moiety comprises the amino acid sequence set forth in SEQ ID NO: 34, or a variant comprising an amino acid sequence having at least about 80%sequence identity
  • the V L of the second binding moiety comprises the amino acid sequence of SEQ ID NO: 35, or a variant comprising an amino acid sequence having at least about 80%sequence identity.
  • the antibody moiety of the second binding moiety comprises an amino acid sequence of SEQ ID NO: 25, or a variant comprising an amino acid sequence having at least about 80%sequence identity.
  • first binding moiety and the second binding moiety are linked in tandem.
  • each of the binding moiety the V H and the V L are fused via a first linker.
  • the surface molecule comprises, from N terminal to C terminal, the first binding moiety, the second binding moiety and the membrane domain.
  • the surface molecule comprises, from N terminal to C terminal, the second binding moiety, the first binding moiety and the membrane domain.
  • the first linker between the V H and the V L is a peptide linker.
  • the first linker between the V H and the V L comprises the amino acid sequence of SEQ ID NO: 9.
  • the first binding moiety and the second binding moiety are independently selected from the group consisting of a sdAb, a scFv, a Fab’, a (Fab’) 2, an Fv, and a peptide ligand.
  • both the first binding moiety and the second binding moiety are scFvs.
  • the GPI attachment signal sequence comprises an amino acid sequence of SEQ ID NO: 39.
  • the first binding moiety and the second binding moiety are fused via a second linker.
  • the second linker between the first binding moiety and the second binding moiety is a peptide linker.
  • the second linker between the first binding moiety and the second binding moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9-16 and 45.
  • the second linker between the first binding moiety and the second binding moiety comprises the amino acid sequence of SEQ ID NO: 9 or 45.
  • the surface molecule comprises the amino acid sequence of SEQ ID NO: 52 or 53.
  • the first binding moiety or the second binding moiety is fused to N-terminus of the membrane domain via a third linker.
  • the third linker is a peptide linker.
  • the third linker is selected from the group consisting of SEQ ID Nos: 9-24, 40 and 46-49.
  • the third linker further comprises a tag.
  • the tag is a HA tag comprises an amino acid sequence of SEQ ID NO: 41.
  • the surface molecule comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 36-37 and 54-57.
  • the surface molecule further comprises a signal peptide at the N-terminus of the molecule that promotes the tethering of the surface molecule to the membrane.
  • the signal peptide is a CD8 ⁇ signal peptide.
  • the cell confers herd immunity against HIV.
  • the cell is a stem cell.
  • the cell is an embryonic stem cell, a hematopoietic stem cell, a mesenchymal stem cell or an induced pluripotent stem cell.
  • the present application provides an engineered cell expressing a surface molecule, wherein the surface molecule comprises:
  • a second binding moiety that specifically binds to an HIV antigen competitively with an anti-HIV antibody, or binds to the same epitope as that of the anti-HIV antibody and prevents HIV from infecting the engineered cell;
  • a membrane domain that tethers the molecule to the membrane of the engineered cell or facilitates the tethering of the surface molecule to the membrane of the engineered cell, wherein the membrane domain comprises a Glycosylphosphatidylinositol (GPI) attachment signal sequence.
  • GPI Glycosylphosphatidylinositol
  • the T cell surface antigen is CCR5.
  • the anti-HIV antibody is 10E8.
  • the first binding moiety specifically binds to CCR5 competitively with C1-13 or C1-14, or specifically binds to the same epitope as that of C1-13 or C1-14.
  • the first binding moiety comprises an antibody moiety comprising a heavy chain variable region (V H ) and a light chain variable region (V L )
  • V H comprises a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 3, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 5
  • the V L comprises a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8.
  • the V H of the first binding moiety comprises the amino acid sequence of SEQ ID NO: 32 or a variant comprising an amino acid sequence having at least about 80%sequence identity
  • the V L of the first binding moiety comprises the amino acid sequence of SEQ ID NO: 33 or a variant comprising an amino acid sequence having at least about 80%sequence identity.
  • the V H in the first binding moiety is fused to the N-terminus of the V L . In other embodiments, in the first binding moiety the V H is fused to the C-terminus of the V L .
  • the antibody moiety of the first binding moiety comprises an amino acid sequence of SEQ ID NO: 1 or 2, or a variant comprising an amino acid sequence having at least about 80%sequence identity.
  • the second binding moiety comprises an antibody moiety comprising a heavy chain variable region (V H ) and a light chain variable region (V L )
  • V H comprises a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a HC- CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 28
  • V L comprises a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 29, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 31.
  • the V H in the second binding moiety is fused to the N-terminus of the V L . In other embodiments, in the second binding moiety the V H is fused to the C-terminus of the V L .
  • the V H of the second binding moiety comprises the amino acid sequence set forth in SEQ ID NO: 34, or a variant comprising an amino acid sequence having at least about 80%sequence identity
  • the V L of the second binding moiety comprises the amino acid sequence of SEQ ID NO: 35, or a variant comprising an amino acid sequence having at least about 80%sequence identity.
  • the antibody moiety of the second binding moiety comprises an amino acid sequence of SEQ ID NO: 25, or a variant comprising an amino acid sequence having at least about 80%sequence identity.
  • first binding moiety and the second binding moiety are linked in tandem.
  • the surface molecule comprises, from N terminal to C terminal, the first binding moiety, the second binding moiety and the membrane domain.
  • the surface molecule comprises, from N terminal to C terminal, the second binding moiety, the first binding moiety and the membrane domain.
  • each of the binding moiety the V H and the V L are fused via a first linker.
  • the first linker between the V H and the V L is a peptide linker.
  • the first linker between the V H and the V L comprises the amino acid sequence of SEQ ID NO: 9.
  • the V H is fused to the N-terminus of the V L .
  • the V H is fused to the C-terminus of the V L .
  • the first binding moiety and the second binding moiety are independently selected from the group consisting of a sdAb, a scFv, a Fab’, a (Fab’) 2, an Fv, and a peptide ligand.
  • both the first binding moiety and the second binding moiety are scFvs.
  • the surface molecule comprises, from N terminal to C terminal, the V H of the first binding moiety, the V L of the first binding moiety, the V H of the second binding moiety and the V L of the second binding moiety. In some embodiments, the surface molecule comprises, from N terminal to C terminal, the V L of the first binding moiety, the V H of the first binding moiety, the V H of the second binding moiety and the V L of the second binding moiety. In some embodiments, the surface molecule comprises, from N terminal to C terminal, the V H of the first binding moiety, the V L of the first binding moiety, the V L of the second binding moiety and the V H of the second binding moiety.
  • the surface molecule comprises, from N terminal to C terminal, the V L of the first binding moiety, the V H of the first binding moiety, the V L of the second binding moiety and the V H of the second binding moiety. In some embodiments, the surface molecule comprises, from N terminal to C terminal, the V H of the second binding moiety, the V L of the second binding moiety, the V H of the first binding moiety and the V L of the first binding moiety. In some embodiments, the surface molecule comprises, from N terminal to C terminal, the V L of the second binding moiety, the V H of the second binding moiety, the V H of the first binding moiety and the V L of the first binding moiety.
  • the surface molecule comprises, from N terminal to C terminal, the V H of the second binding moiety, the V L of the second binding moiety, the V L of the first binding moiety and the V H of the first binding moiety. In some embodiments, the surface molecule comprises, from N terminal to C terminal, the V L of the second binding moiety, the V H of the second binding moiety, the V L of the first binding moiety and the V H of the first binding moiety.
  • the first binding moiety and the second binding moiety are fused via a second linker.
  • the second linker is a peptide linker.
  • the second linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9-16 and 45.
  • the second linker comprises the amino acid sequence of SEQ ID NO: 9 or 45.
  • the surface molecule comprises the amino acid sequence of SEQ ID NO: 52 or 53.
  • the GPI attachment signal sequence comprises the amino acid sequence of SEQ ID NO: 39.
  • the first binding moiety or the second binding moiety is fused to N-terminus of the membrane domain via a third linker.
  • the third linker is a peptide linker.
  • the third linker is selected from the group consisting of SEQ ID Nos: 9-24, 40 and 46-49.
  • the third linker further comprises a tag; preferably, the tag is an HA tag comprising an amino acid sequence of SEQ ID NO: 41.
  • the surface molecule comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 36-37 and 54-57.
  • the surface molecule further comprises a signal peptide at the N-terminus of the molecule that promotes the tethering of the surface molecule to the membrane.
  • the signal peptide is a CD8 ⁇ signal peptide.
  • the engineered cell is a stem cell.
  • the stem cell is an embryonic stem cell (ESC) . In some embodiments, the stem cell is hematopoietic stem cell (HSC) . In some embodiments, the stem cell is a mesenchymal stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell (iPSC) .
  • ESC embryonic stem cell
  • HSC hematopoietic stem cell
  • iPSC induced pluripotent stem cell
  • the engineered cell is an immune cell.
  • the engineered cell is a T cell or a Natural Killer cell.
  • the present application provides a plurality of the engineered cells described above, wherein upon a) mixing with a plurality of cells not expressing the surface molecule and being susceptible to HIV infection; and b) contacting of the mixture obtained in a) with an HIV, the percentage of cells not infected with the HIV is at least about 10%higher than the percentage of the engineered cells in the mixture.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the engineered cell or the plurality of engineered cells described above, and a pharmaceutically acceptable carrier.
  • the present application provides a method of preparing the engineered cell described above, comprising introducing one or more nucleic acids encoding the surface molecule into a cell, thereby obtaining the engineered cell.
  • the method further comprises selecting the engineered cell based on its expression of the surface molecule.
  • the engineered cell is a stem cell or an immune cell.
  • the present application provides a method of enriching engineered cell described above, comprising selecting the engineered cell based upon the first binding moiety or the second binding moiety.
  • the engineered cell is a stem cell or an immune cell.
  • the present application provides a method of treating an individual infected with HIV, comprising administering to the individual an effective amount of the engineered cell or the pharmaceutical composition described above.
  • the engineered cell is autologous or allogeneic to the individual.
  • At least about 5%of the T cells in the individual express the surface molecule after administration of the engineered cell or the pharmaceutical composition.
  • At least about 20%of the T cells in the individual are resistant to HIV infection after administration of the engineered cell or the pharmaceutical composition.
  • the method further comprises administering a second therapy.
  • FIG. 1 depicts the GFP expression of TZM-bl cells transduced with GPI-scFv constructs.
  • FIG. 2 depicts blocking effects of TZM-bl cells transduced with an exemplary GPI-scFv construct and TZM-bl cells not transduced with a GPI-scFv construct in mixture against HIV pseudovirus.
  • FIG. 3 depicts the GFP expression of TZM-bl cells transduced with GPI-scFv constructs (GPI-604) .
  • FIG. 4 depicts blocking effects of engineered TZM-bl cells (GPI-604 clone 2 and clone 6) .
  • the present application provides engineered cells (such as stem cells, immune cells) comprising a nucleic acid encoding a surface molecule comprising: a) a first binding moiety that specifically binds to a T cell surface antigen and prevents the binding of the T cell surface antigen to its cognitive ligand on HIV and b) a second binding moiety that specifically binds to an HIV antigen and prevents the binding of the HIV antigen to the engineered cell.
  • the surface molecule further comprises a membrane domain (e.g., a GPI attachment sequence) that tethers the binding moiety to the cell membrane or facilitates the tethering of the molecule to the membrane.
  • the engineered cell achieves resistance to HIV infection.
  • the first binding moiety specifically binds to CCR5.
  • the second binding moiety binds to an HIV antigen competitively with 10E8, or binds to the same epitope as that of 10E8.
  • compositions comprising a plurality of the engineered cell described herein, pharmaceutical compositions comprising the engineered cells, methods of preparing the engineered cells, methods of treating an individual infected with HIV by administering the engineered cells.
  • the present application in one aspect provides a solution to the long-lasting challenges in gene-editing based stem cell therapy for patients infected with HIV.
  • bi-allelic CCR5 knockout HSCs are HIV-resistant and can be a promising therapy.
  • they are difficult to screen or enrich due to the lack of a selection marker.
  • Selection marker cannot be integrated in ribonucleoprotein (RNP) under currently available genome-editing tools (such as CRISPR-Cas9) .
  • CRISPR-Cas9 ribonucleoprotein
  • high concentrations of RNP may lead to high off-target site cleavage.
  • a highly pure HIV-resistant cell population can be obtained via enrichment based upon the surface molecule.
  • This approach provides a feasible solution to the challenges for gene-editing based stem cell therapy, and great therapeutic benefits to treating HIV patients.
  • engineered cells with certain surface molecules confer a cell-level herd immunity phenomenon.
  • the engineered cells not only exhibit the resistance against HIV infection, but also confer immunity to other cells that are originally susceptible to HIV infection.
  • engineered cells expressing exemplary surface molecules such surface molecules that have a) an anti-CCR5 binding moiety, b) an anti-HIV binding moiety, and c) a membrane domain
  • antibody is used in its broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) , full-length antibodies and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity.
  • antibody includes conventional four-chain antibodies, and single-domain antibodies, such as heavy-chain only antibodies or fragments thereof, e.g., V H H.
  • a full-length four-chain antibody comprises two heavy chains and two light chains.
  • the variable regions of the light and heavy chains are responsible for antigen binding.
  • the variable domains of the heavy chain and light chain may be referred to as “V H ” and “V L ” , respectively.
  • the variable regions in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light chain (LC) CDRs including LC-CDR1, LC-CDR2, and LC-CDR3, heavy chain (HC) CDRs including HC-CDR1, HC-CDR2, and HC-CDR3) .
  • CDRs complementarity determining regions
  • CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani, 1997, J. Mol. Biol., 273: 927-948; Chothia 1985, J. Mol Biol., 186: 651-663; Chothia 1987, J. Mol. Biol., 196: 901-917; Chothia 1989, Nature, 342: 877-883; Kabat 1987, Sequences of Proteins of Immunological Interest, Fourth Edition. US Govt. Printing Off. No. 165-492; Kabat 1991, Sequences of Proteins of Immunological Interest, Fifth Edition. NIH Publication No. 91-3242) .
  • the three CDRs of the heavy or light chains are interposed between flanking stretches known as framework regions (FRs) , which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops.
  • the constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions.
  • Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain.
  • the five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ heavy chains, respectively.
  • lgG1 ⁇ 1 heavy chain
  • lgG2 ⁇ 2 heavy chain
  • lgG3 ⁇ 3 heavy chain
  • lgG4 ⁇ 4 heavy chain
  • lgA1 ⁇ 1 heavy chain
  • lgA2 ⁇ 2 heavy chain
  • HCAb heavy chain-only antibody
  • HCAb refers to a functional antibody, which comprises heavy chains, but lacks the light chains usually found in 4-chain antibodies.
  • Camelid animals (such as camels, llamas, or alpacas) are known to produce HCAbs.
  • single-domain antibody refers to a single antigen-binding polypeptide having three complementary determining regions (CDRs) .
  • CDRs complementary determining regions
  • the sdAb alone is capable of binding to the antigen without pairing with a corresponding CDR-containing polypeptide.
  • single-domain antibodies are engineered from camelid HCAbs, and their heavy chain variable domains are referred herein as “V H Hs” (Variable domain of the heavy chain of the Heavy chain antibody) .
  • Camelid sdAb is one of the smallest known antigen-binding antibody fragments (see, e.g., Hamers-Casterman et al., Nature 363: 446-8 (1993) ; Greenberg et al., Nature 374: 168-73 (1995) ; Hassanzadeh-Ghassabeh et al., Nanomedicine (Lond) , 8: 1013-26 (2013) ) .
  • a basic V H H has the following structure from the N-terminus to the C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3.
  • antibody moiety includes full-length antibodies and antigen-binding fragments thereof.
  • a full-length antibody comprises two heavy chains and two light chains.
  • the variable regions of the light and heavy chains are responsible for antigen binding.
  • the variable regions in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light chain (LC) CDRs including LC-CDR1, LC-CDR2, and LC-CDR3, heavy chain (HC) CDRs including HC-CDR1, HC-CDR2, and HC-CDR3) .
  • CDRs complementarity determining regions
  • CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991) .
  • the three CDRs of the heavy or light chains are interposed between flanking stretches known as framework regions (FRs) , which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops.
  • FRs framework regions
  • the constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions.
  • Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain.
  • the five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ heavy chains, respectively.
  • Several of the major antibody classes are divided into subclasses such as lgG1 ( ⁇ 1 heavy chain) , lgG2 ( ⁇ 2 heavy chain) , lgG3 ( ⁇ 3 heavy chain) , lgG4 ( ⁇ 4 heavy chain) , lgA1 ( ⁇ 1 heavy chain) , or lgA2 ( ⁇ 2 heavy chain) .
  • antigen-binding fragment refers to an antibody fragment including, for example, a diabody, a Fab, a Fab’, a F (ab’) 2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv) , a (dsFv) 2, a bispecific dsFv (dsFv-dsFv’) , a disulfide stabilized diabody (ds diabody) , a single-chain Fv (scFv) , an scFv dimer (bivalent diabody) , a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure.
  • an antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment (e.g., a parent scFv) binds.
  • an antigen-binding fragment may comprise one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies.
  • “Fv” is the minimum antibody fragment, which contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy-and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the heavy and light chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • Single-chain Fv also abbreviated as “sFv” or “scFv, ” are antibody fragments that comprise the V H and V L antibody domains connected into a single polypeptide chain.
  • the scFv polypeptide further comprises a polypeptide linker between the V H and V L domains, which enables the scFv to form the desired structure for antigen binding.
  • Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994) .
  • CDR complementarity determining region
  • CDR complementarity determining region
  • Residue numbering follows the nomenclature of Kabat et al., supra 2 Residue numbering follows the nomenclature of Chothia et al., supra 3 Residue numbering follows the nomenclature of MacCallum et al., supra 4 Residue numbering follows the nomenclature of Lefranc et al., supra 5 Residue numbering follows the nomenclature of Honegger and Plückthun, supra
  • Framework or “FR” residues are those variable-domain residues other than the CDR residues as herein defined.
  • a monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies.
  • such a monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences.
  • the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones.
  • a selected target binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of this invention.
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the invention may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein, Nature 256: 495-97 (1975) ; Hongo et al., Hybridoma 14 (3) : 253-260 (1995) , Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.
  • the monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain (s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984) ) .
  • Chimeric antibodies include antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with the antigen of interest.
  • “Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a HVR of the recipient are replaced by residues from a HVR of a non-human species (donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and/or capacity.
  • donor antibody such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and/or capacity.
  • FR residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc) , typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • a “human antibody” is one that possesses an amino acid sequence, which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • Human antibodies can be produced using various techniques known in the art, including phage-display libraries. Hoogenboom and Winter, J. Mol. Biol. 227: 381 (1991) ; Marks et al., J. Mol. Biol. 222: 581 (1991) . Also available for the preparation of human monoclonal antibodies are methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R.
  • Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSETM technology) . See also, for example, Li et al., Proc. Natl. Acad. Sci. USA 103: 3557-3562 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.
  • the term “binds” “specifically binds to” or is “specific for” refers to measurable and reproducible interactions such as binding between a target and an antibody, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules.
  • an antibody that binds to or specifically binds to a target (which can be an epitope) is an antibody that binds this target with greater affinity, avidity, more readily, and/or with greater duration than it binds to other targets.
  • the extent of binding of an antibody to an unrelated target is less than about 10%of the binding of the antibody to the target as measured, e.g., by a radioimmunoassay (RIA) .
  • RIA radioimmunoassay
  • an antibody that specifically binds to a target has a dissociation constant (Kd) of ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, or ⁇ 0.1 nM.
  • Kd dissociation constant
  • an antibody specifically binds to an epitope on a protein that is conserved among the protein from different species.
  • specific binding can include, but does not require exclusive binding.
  • the term “specificity” refers to selective recognition of an antigen binding protein (such as a chimeric receptor or an antibody construct) for a particular epitope of an antigen. Natural antibodies, for example, are monospecific.
  • the term “multispecific” as used herein denotes that an antigen binding protein has two or more antigen-binding sites of which at least two bind different antigens or epitopes.
  • Bispecific as used herein denotes that an antigen binding protein has two different antigen-binding specificities.
  • the term “monospecific” as used herein denotes an antigen binding protein that has one or more binding sites each of which bind the same antigen or epitope.
  • valent denotes the presence of a specified number of binding sites in an antigen binding protein.
  • a natural antibody for example or a full-length antibody has two binding sites and is bivalent.
  • trivalent tetravalent
  • pentavalent hexavalent
  • Percent (%) amino acid sequence identity with respect to a polypeptide sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN TM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared.
  • polypeptides having at least 70%, 85%, 90%, 95%, 98%or 99%identity to specific polypeptides described herein and preferably exhibiting substantially the same functions, as well as polynucleotide encoding such polypeptides, are contemplated.
  • recombinant refers to a biomolecule, e.g., a gene or protein, that (1) has been removed from its naturally occurring environment, (2) is not associated with all or a portion of a polynucleotide in which the gene is found in nature, (3) is operatively linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature.
  • the term “recombinant” can be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs that are biologically synthesized by heterologous systems, as well as proteins and/or mRNAs encoded by such nucleic acids.
  • express refers to translation of a nucleic acid into a protein. Proteins may be expressed and remain intracellular, become a component of the cell surface membrane, or be secreted into extracellular matrix or medium.
  • host cell refers to a cell that can support the replication or expression of the expression vector.
  • Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells, such as yeast, insect cells, amphibian cells, or mammalian cells.
  • transfected or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a “transfected” or “transformed” or “transduced” cell is one that has been transfected, transformed or transduced with exogenous nucleic acid.
  • in vivo refers to inside the body of the organism from which the cell is obtained. “Ex vivo” or “in vitro” means outside the body of the organism from which the cell is obtained.
  • cell includes the primary subject cell and its progeny.
  • autologous refers to any material derived from the same individual to whom it is later to be re-introduced into the individual.
  • Allogeneic refers to a graft derived from a different individual of the same species.
  • domain when referring to a portion of a protein is meant to include structurally and/or functionally related portions of one or more polypeptides that make up the protein.
  • a transmembrane domain of an immune cell receptor may refer to the portions of each polypeptide chain of the receptor that span the membrane.
  • a domain may also refer to related portions of a single polypeptide chain.
  • a transmembrane domain of a monomeric receptor may refer to portions of the single polypeptide chain of the receptor that span the membrane.
  • a domain may also include only a single portion of a polypeptide.
  • isolated nucleic acid as used herein is intended to mean a nucleic acid of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the “isolated nucleic acid” (1) is not associated with all or a portion of a polynucleotide in which the “isolated nucleic acid” is found in nature, (2) is operably linked to a polynucleotide which it is not linked to in nature, or (3) does not occur in nature as part of a larger sequence.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • the phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron (s) .
  • operably linked refers to functional linkage between a regulatory sequence and a nucleic acid sequence resulting in expression of the latter.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • inducible promoter refers to a promoter whose activity can be regulated by adding or removing one or more specific signals.
  • an inducible promoter may activate transcription of an operably linked nucleic acid under a specific set of conditions, e.g., in the presence of an inducing agent or conditions that activates the promoter and/or relieves repression of the promoter.
  • treatment is an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease) , preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing or improving the quality of life, increasing weight gain, and/or prolonging survival.
  • treatment is a reduction of pathological consequence of the disease (such as, for example, tumor volume in cancer) .
  • the methods of the invention contemplate any one or more of
  • pharmaceutically acceptable or “pharmacologically compatible” is meant a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained.
  • Pharmaceutically acceptable carriers or excipients have preferably met the required standards of toxicological and manufacturing testing and/or are included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug administration.
  • Administration “in combination with” one or more further agents includes simultaneous and sequential administration in any order.
  • the term “simultaneously” is used herein to refer to administration of two or more therapeutic agents, where at least part of the administration overlaps in time or where the administration of one therapeutic agent falls within a short period of time relative to administration of the other therapeutic agent.
  • the two or more therapeutic agents are administered with a time separation of no more than about 15 minutes, such as no more than about any of 10, 5, or 1 minute.
  • administration of two or more therapeutic agents where the administration of one or more therapeutic agent (s) continues after discontinuing the administration of one or more other agent (s) .
  • administration of the two or more agents are administered with a time separation of more than about 15 minutes, such as about any of 20, 30, 40, 50, or 60 minutes, 1 day, 2 days, 3 days, 1 week, 2 weeks, or 1 month, or longer.
  • a “subject” or an “individual” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.
  • references to "about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to "about X” includes description of "X” .
  • reference to "not" a value or parameter generally means and describes "other than” a value or parameter.
  • the method is not used to treat cancer of type X means the method is used to treat cancer of types other than X.
  • a and/or B is intended to include both A and B; A or B; A (alone) ; and B (alone) .
  • the term “and/or” as used herein a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone) ; B (alone) ; and C (alone) .
  • Engineered cell comprising a nucleic acid encoding a surface molecule
  • the present application in one aspect provides an engineered cell comprising a nucleic acid encoding a surface molecule comprising a) a first binding moiety that specifically binds to a T cell surface antigen and prevents the binding of the T cell surface antigen to its cognitive ligand on HIV, b) a second binding moiety that specifically binds to an HIV antigen, and c) a membrane domain that tethers the molecule to the membrane or facilitates the tethering of the molecule to the membrane, wherein the membrane domain comprises a Glycosylphosphatidylinositol (GPI) attachment signal sequence.
  • the T cell surface antigen is CCR5.
  • the anti-HIV antibody is 10E8.
  • the GPI attachment signal sequence comprises the amino acid sequence of SEQ ID NO: 39.
  • an engineered stem cell e.g., hematopoietic stem cell (HSC)
  • HSC hematopoietic stem cell
  • a first binding moiety that specifically binds to CCR5
  • a second binding moiety that specifically binds to an HIV antigen
  • a membrane domain comprising a GPI attachment signal sequence (e.g., SEQ ID NO: 39)
  • the first binding moiety specifically binds to CCR5 competitively with C1-13 or C1-14, or specifically binds to the same epitope as that of C1-13 or C1-14.
  • the first binding moiety comprises an antibody moiety comprising a heavy chain variable region (V H ) and a light chain variable region (V L )
  • V H comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 3
  • the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4
  • the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 5
  • the V L comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8.
  • the antibody moiety comprises a) the VH comprising the amino acid sequence set forth in SEQ ID NO: 32 or a variant comprising an amino acid sequence having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity, and b) the VL comprising the amino acid sequence of SEQ ID NO: 33 or a variant comprising an amino acid sequence having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.
  • the second binding moiety specifically binds to an HIV antigen competitively with 10E8, or binds to the same epitope as that of 10E8 and prevents the binding of the HIV antigen to the engineered cell.
  • the second binding moiety comprises an antibody moiety comprising a heavy chain variable region (V H ) and a light chain variable region (V L ) , wherein the V H comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 26, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 28, and the V L comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 29, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 31.
  • the antibody moiety comprises a) the VH comprising the amino acid sequence set forth in SEQ ID NO: 34, or a variant comprising an amino acid sequence having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity, and b) the VL comprising the amino acid sequence of SEQ ID NO: 35, or a variant comprising an amino acid sequence having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.
  • an engineered stem cell e.g., hematopoietic stem cell (HSC)
  • HSC hematopoietic stem cell
  • a first binding moiety comprising an scFv that specifically binds to CCR5
  • the scFv comprises the amino acid sequence of SEQ ID NO: 1 or 2, or a variant comprising an amino acid sequence having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity
  • b) a second binding moiety comprising an scFv that specifically binds to an HIV antigen wherein the second binding moiety comprises the amino acid sequences of SEQ ID NO: 25, or a variant comprising an amino acid sequence having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity
  • a membrane domain comprising a GPI attachment signal sequence
  • the V H and the V L described herein are fused via a linker.
  • the linker between the V H and the V L is a peptide linker.
  • the linker between the V H and the V L comprises the amino acid sequence of SEQ ID NO: 9.
  • the binding moiety is a sdAb, a scFv, a Fab’, a (Fab’) 2 , an Fv, or a peptide ligand.
  • the binding moiety is a scFv.
  • the first binding moiety and the second binding moiety are fused via a second linker.
  • the second linker between a) the first binding moiety and b) the second binding moiety comprises a peptide linker.
  • the second linker is selected from the group consisting of SEQ ID NOs: 9-16 and 45.
  • the first or second binding moiety is fused to N-terminus of the membrane domain via a third linker.
  • the third linker between a) the binding moiety and b) the membrane domain comprises a peptide linker.
  • the third linker is selected from the group consisting of SEQ ID NOs: 9-16, 40 and 46-49.
  • the third linker further comprises a tag; preferably, the tag is an HA tag comprises an amino acid sequence of SEQ ID NO: 41.
  • the engineered cell is a stem cell.
  • the stem cell is an embryonic stem cell (ESC) .
  • the stem cell is hematopoietic stem cell (HSC) .
  • the stem cell is a mesenchymal stem cell.
  • the stem cell is an induced pluripotent stem cell (iPSC) .
  • the engineered cell is an immune cell.
  • the immune cell is a T cells.
  • the immune cell is a B cell.
  • the immune cell is a Natural killer cell (NK cell) .
  • the surface molecule comprises: a) a first binding moiety that specifically binds to a T cell surface antigen and prevents the binding of the T cell surface antigen to its cognitive ligand on HIV; b) a second binding moiety that specifically binds to an HIV antigen competitively with an anti-HIV antibody, or binds to the same epitope as that of the anti-HIV antibody; and c) a membrane domain that can tether the surface molecule to a cell membrane or facilitate the tethering of the surface molecule to the cell membrane after being expressed in a cell, wherein the membrane domain comprises a Glycosylphosphatidylinositol (GPI) attachment signal sequence.
  • GPI Glycosylphosphatidylinositol
  • the T cell surface antigen is CCR5.
  • the first binding moiety specifically binds to CCR5 competitively with C1-13 or C1-14, or specifically binds to the same epitope as that of C1-13 or C1-14.
  • the first binding moiety comprises an antibody moiety comprising a heavy chain variable region (V H ) and a light chain variable region (V L )
  • V H comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 3
  • the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4
  • the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 5
  • the V L comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8.
  • the first binding moiety comprises the amino acid sequence of SEQ ID NO: 1 or 2, or a variant comprising an amino acid sequence having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.
  • the anti-HIV antibody is 10E8.
  • the second binding moiety comprises an antibody moiety comprising a heavy chain variable region (V H ) and a light chain variable region (V L )
  • V H comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 26, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 28,
  • V L comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 29, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 31.
  • the binding moiety comprises the amino acid sequences of SEQ ID NO: 25, or a variant comprising an amino acid sequence having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.
  • the GPI attachment signal sequence comprises the amino acid sequence of SEQ ID NO: 39.
  • the surface molecule comprises, from N terminal to C terminal, the first binding moiety, the second binding moiety and the membrane domain.
  • the surface molecule comprises the amino acid sequence of SEQ ID NO: 36, 54 or 55.
  • the surface molecule comprises, from N terminal to C terminal, the second binding moiety, the first binding moiety and the membrane domain.
  • the surface molecule comprises the amino acid sequence of SEQ ID NO: 37, 56 or 57.
  • the present application in another aspect provides an engineered cell expressing a surface molecule described above, and exhibiting herd immunity against HIV.
  • Herd immunity against HIV described herein refers to the phenomena that a cell expressing a certain surface molecule that prevents or inhibits the infection of HIV (such as any of the surface molecules described herein) is not only resistant to HIV infection itself, but also confers the anti-HIV immunity to another cell that is originally susceptible to HIV infection.
  • Herd immunity against HIV can be tested by mixing a plurality of cells expressing the surface molecule with a plurality of cells not expressing the surface molecule and are susceptible to HIV infection at a certain ratio (such as 4: 1, 3: 1, 2: 1, 1: 1, 1: 2, 1: 3, 1: 4) , and then incubating the mixture of the cells with HIV.
  • Herd immunity exists when the number of cells resistant to HIV infection after incubation is higher than (such as at least about 5%, 10%, 15%, or 20%higher than) the number of cells expressing the surface molecule. See e.g., Examples 2 for exemplary illustration of methods that can be used to test herd immunity effect.
  • the cell is a stem cell (such as a hematopoietic stem cell (HSC) ) .
  • the cell is an immune cell.
  • the cell is a T cell.
  • a plurality of the engineered cell (such as a plurality of any of the engineered cell described herein) , wherein upon a) mixture with a plurality of cells not expressing the surface molecule and are susceptible to HIV infection and b) contact of the cellular composition with an HIV, the percentage of cells not infected with the HIV is higher (such as at least about 10%higher) than the percentage of engineered cells.
  • the number of the plurality of cells not comprising a nucleic acid expressing the surface molecules between about 80%to about 120% (such as about 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%or 120%) of number of the plurality of engineered cells.
  • the plurality of cells not comprising a nucleic acid expressing the surface molecule express CCR5 and are susceptible to HIV.
  • a population of stem cells comprising a plurality of engineered stem cells (e.g., engineered hematopoietic stem cells) such as any of those described above, wherein purity of the engineered stem cell in the population of the stem cells is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.
  • the engineered stem cells express an anti-CCR5 antibody moiety (e.g., an anti-CCR5 scFv) and an anti-HIV antibody moiety (e.g., an anti-HIV scFv, e.g., SEQ ID NO: 25) .
  • an anti-CCR5 antibody moiety e.g., an anti-CCR5 scFv
  • an anti-HIV antibody moiety e.g., an anti-HIV scFv, e.g., SEQ ID NO: 25
  • the surface molecule comprises a) a first binding moiety that specifically binds to a T cell surface antigen (e.g., CCR5) and prevents the binding of the T cell surface antigen to its cognitive ligand on HIV; b) a second binding moiety that specifically binds to an HIV antigen competitively with an anti-HIV antibody, or binds to the same epitope as that of the anti-HIV antibody; and c) a membrane domain that can tether the surface molecule to a cell membrane or facilitate the tethering of the surface molecule to the cell membrane after being expressed in a cell.
  • a T cell surface antigen e.g., CCR5
  • a second binding moiety that specifically binds to an HIV antigen competitively with an anti-HIV antibody, or binds to the same epitope as that of the anti-HIV antibody
  • a membrane domain that can tether the surface molecule to a cell membrane or facilitate the tethering of the surface molecule to the cell membrane after
  • the cell after the surface molecule is expressed by a cell, the cell confers herd immunity against HIV. In some embodiments, the cell expresses CCR5 and 10E8. In some embodiments, the cell is a TZM-bl cell.
  • the binding moiety (e.g, the first binding moiety or the second binding moiety) is fused to N-terminus of the membrane domain. In some embodiments, the binding moiety is fused to N-terminus of the membrane domain via a linker (such as any of the linkers described in the “linker” section) . In some embodiments, the linker between a) the binding moiety and b) the membrane domain comprises a peptide linker. In some embodiments, the linker is selected from the group consisting of SEQ ID NOs: 9-16, 40 and 46-49. In some embodiments, the binding moiety is fused to N-terminus of the membrane domain without a linker.
  • the surface molecule comprises, from N terminal to C terminal, the first binding moiety, the second binding moiety and the membrane domain. In some embodiments, the surface molecule comprises the amino acid sequence of SEQ ID NO: 36, 54, or 55. In some embodiments, the surface molecule comprises, from N terminal to C terminal, the second binding moiety, the first binding moiety and the membrane domain. In some embodiments, the surface molecule comprises the amino acid sequence of SEQ ID NO: 37, 56 or 57.
  • the binding moiety (e.g, the first binding moiety or the second binding moiety) is directly fused to N-terminus of the membrane domain. That is, there is not a linker between the binding moiety (e.g, the first binding moiety or the second binding moiety) and the membrane domain (e.g., a GPI attachment signal sequence) .
  • the surface molecule may comprise the amino acid sequence of SEQ ID NO: 52 or 53.
  • the surface molecule further comprises a signal peptide at the N-terminus of the molecule that promotes the tethering of the surface molecule to the membrane.
  • the signal peptide is a CD8 ⁇ signal peptide (e.g., SEQ ID NO: 38) .
  • the binding moieties described in this application are a) a first binding moiety that specifically binds to a T cell surface antigen and prevents the binding of the T cell surface antigen to its cognitive ligand on HIV, and b) a second binding moiety that specifically binds to an HIV antigen and prevents the binding of the HIV antigen to the engineered cell.
  • the T cell surface antigen is CCR5.
  • the second binding moiety specifically binds to an HIV antigen competitively with 10E8, or binds to the same epitope as that of 10E8.
  • the binding moiety is an antibody moiety that has a heavy chain variable region (V H ) and a light chain variable region (V L ) , wherein the V H comprises a HC-CDR1, a HC-CDR2, and a HC-CDR3, and the V L comprises a LC-CDR1, a LC-CDR2, and a LC-CDR3.
  • V H heavy chain variable region
  • V L light chain variable region
  • the V H and the V L are fused via a linker (such as any of the linkers described in the “linker” section) .
  • the linker between the V H and the V L is a peptide linker.
  • the linker between the V H and the V L comprises the amino acid sequence of SEQ ID NO: 9.
  • the binding moiety is a sdAb, a scFv, a Fab’, a (Fab’) 2 , an Fv, or a peptide ligand.
  • the binding moiety is a scFv comprising a heavy chain variable region (V H ) and a light chain variable region (V L ) .
  • V H is fused to the N-terminus of the V L .
  • the V H is fused to the C-terminus of the V L .
  • the V H and the V L are fused via a linker (such as any of the linkers described in the “linker” section) .
  • the linker between the V H and the V L is a peptide linker.
  • the linker between the V H and the V L comprises the amino acid sequence of SEQ ID NO: 9.
  • binding moieties are described below.
  • the T cell surface antigen is CCR5.
  • the binding moiety comprises an anti-CCR5 antibody moiety.
  • the binding moiety specifically binds to CCR5 competitively with C1-13 or C1-14, or specifically binds to the same epitope as that of C1-13 or C1-14.
  • the first binding moiety specifically binds to CCR5 competitively with C1-13, or specifically binds to the same epitope as that of C1-13.
  • the first binding moiety comprises an antibody moiety comprising a heavy chain variable region (V H ) and a light chain variable region (V L ) , wherein the V H comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 3, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 5, and the V L comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8.
  • the binding moiety comprises the amino acid sequence of SEQ ID NO: 1, or a variant comprising an amino acid sequence having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.
  • the second binding moiety specifically binds to an HIV antigen (e.g., an anti-HIV antibody) .
  • an HIV antigen e.g., an anti-HIV antibody
  • the second binding moiety specifically binds to HIV competitively with 10E8, or specifically binds to the same epitope as that of 10E8.
  • the binding moiety comprises an antibody moiety comprising a heavy chain variable region (V H ) and a light chain variable region (V L ) , wherein the V H comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 26, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 28, and the V L comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 29, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 31.
  • the binding moiety comprises the amino acid sequences of SEQ ID NO: 25, or a variant comprising an amino acid sequence having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.
  • Membrane domains described in this application include any molecule that is capable of a) tethering the binding moiety or the surface molecule described herein to the membrane of a cell or b) facilitating the tethering of the binding moiety or the surface molecule to the membrane.
  • the membrane domain capable of facilitating the tethering of the binding moiety or the surface molecule to the membrane comprises a GPI attachment signal sequence (e.g., SEQ ID NO: 39) .
  • the GPI attachment signal sequence comprises the amino acid sequence of SEQ ID NO: 39.
  • the present application also provide multispecific (e.g., bispecific) surface molecules described herein.
  • the surface molecule described herein comprises two or more moieties (e.g., two or more binding moieties as described herein) .
  • the surface molecule comprises a first binding moiety that specifically binds to a T cell surface antigen (e.g., CCR5) and prevents the binding of the T cell surface antigen to its cognitive ligand on HIV, and a second binding moiety that specifically binds to an HIV antigen and prevents the binding of the HIV antigen to the engineered cell, and wherein the surface molecule further comprises a membrane domain that tethers the molecule to the membrane or facilitates the tethering of the molecule to the membrane.
  • the membrane domain comprises a GPI attachment signal sequence (e.g., SEQ ID NO: 39) .
  • the surface molecule comprises a first binding moiety that specifically binds to CCR5, and a second binding moiety that specifically binds to an HIV antigen and prevents the binding of the HIV antigen to the engineered cell, and wherein the surface molecule further comprises a membrane domain that tethers the molecule to the membrane or facilitates the tethering of the molecule to the membrane.
  • the second binding moiety specifically binds to an HIV antigen competitively with 10E8, or binds to the same epitope as that of 10E8.
  • the membrane domain comprises a GPI attachment signal sequence (e.g., SEQ ID NO: 39) .
  • the first binding moiety specifically binds to CCR5 competitively with C1-13 or C1-14, or specifically binds to the same epitope as that of C1-13 or C1-14.
  • the first binding moiety comprises the amino acid sequence of any one of SEQ ID NOs: 1-2, or a variant comprising an amino acid sequence having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.
  • the second binding moiety specifically binds to an HIV antigen competitively with 10E8, or binds to the same epitope as that of 10E8 and prevents the binding of the HIV antigen to the engineered cell.
  • the second binding moiety comprises the amino acid sequence of SEQ ID NOs: 25, or a variant comprising an amino acid sequence having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.
  • the length, the degree of flexibility and/or other properties of the linker (s) used in the surface molecules may have some influence on properties, including but not limited to the affinity, specificity or avidity for one or more particular antigens or epitopes. For example, longer linkers may be selected to ensure that two adjacent domains do not sterically interfere with one another.
  • a linker (such as peptide linker) comprises flexible residues (such as glycine and serine) so that the adjacent domains are free to move relative to each other.
  • a glycine-serine doublet can be a suitable peptide linker.
  • the linker is a non-peptide linker.
  • the linker is a peptide linker.
  • linker considerations include the effect on physical or pharmacokinetic properties of the resulting compound, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (more or less stable as well as planned degradation) , rigidity, flexibility, immunogenicity, modulation of antibody binding, the ability to be incorporated into a micelle or liposome, and the like.
  • the linker is a peptide linker as described below.
  • the peptide linker has a length of about one to about fifty, about two to about fourth, about three to about thirty, or about four to about twenty amino acids.
  • the linker is a GS linker.
  • the linker comprises an amino acid sequence of any one of SEQ ID NOs: 9-24, 40 and 45-49. In some embodiments, the linker comprises an amino acid sequence of any one of SEQ ID NOs: 9-16 and 23-24. In some embodiments, the linker comprises an amino acid sequence of any one of SEQ ID NOs: 17-22.
  • the peptide linker may have a naturally occurring sequence, or a non-naturally occurring sequence.
  • a sequence derived from the hinge region of heavy chain only antibodies may be used as the linker. See, for example, WO1996/34103.
  • the peptide linker can be of any suitable length. In some embodiments, the peptide linker is at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or more amino acids long. In some embodiments, the peptide linker is no more than about any of 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or fewer amino acids long.
  • the length of the peptide linker is any of about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids long, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, or about 1 amino acid to about 100 amino acids.
  • peptide linker does not comprise any polymerization activity.
  • the characteristics of a peptide linker, which comprise the absence of the promotion of secondary structures, are known in the art and described, e.g., in Dall’Acqua et al. (Biochem. (1998) 37, 9266-9273) , Cheadle et al. (Mol Immunol (1992) 29, 21-30) and Raag and Whitlow (FASEB (1995) 9 (1) , 73-80) .
  • a particularly preferred amino acid in context of the “peptide linker” is Gly.
  • peptide linkers that also do not promote any secondary structures are preferred.
  • the linkage of the domains to each other can be provided by, e.g., genetic engineering.
  • Methods for preparing fused and operatively linked bispecific single chain constructs and expressing them in mammalian cells or bacteria are well-known in the art (e.g. WO 99/54440, Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. 1989 and 1994 or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001) .
  • the peptide linker can be a stable linker, which is not cleavable by proteases, especially by Matrix metalloproteinases (MMPs) .
  • MMPs Matrix metalloproteinases
  • the linker can also be a flexible linker.
  • exemplary flexible linkers include glycine polymers (G) n (SEQ ID NO: 13) , glycine-serine polymers (including, for example, (GS) n (SEQ ID NO: 14) , (GSGGS) n (SEQ ID NO: 15) , (GGGGS) n (SEQ ID NO: 10) , and (GGGS) n (SEQ ID NO: 16, 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 relatively unstructured, and therefore may be able to serve as a neutral tether between components. 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. 11 173-142 (1992) ) .
  • the ordinarily skilled artisan will recognize that design of an antibody fusion protein can include linkers that are all or partially flexible, such that the linker can include a flexible linker portion as well as one or more portions that confer less flexible structure to provide a desired antibody fusion protein structure.
  • the linker is a GS linker.
  • the linker has an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-16 and 23-24.
  • the peptide linker comprises the hinge region of an IgG, such as the hinge region of human IgG1.
  • the linker has a sequence of an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-22.
  • Coupling of two moieties may be accomplished by any chemical reaction that will bind the two molecules so long as both components retain their respective activities.
  • This linkage can include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding and complexation.
  • the binding is covalent binding.
  • Covalent binding can be achieved either by direct condensation of existing side chains or by the incorporation of external bridging molecules.
  • Many bivalent or polyvalent linking agents may be useful in coupling protein molecules in this context.
  • representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzenes and hexamethylene diamines.
  • non-peptide linkers used herein include: (i) EDC (1-ethyl-3- (3-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha- (2-pridyl-dithio) -toluene (Pierce Chem. Co., Cat.
  • linkers described above contain components that have different attributes, thus may lead to bispecific antibodies with differing physio-chemical properties.
  • sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates.
  • NHS-ester containing linkers are less soluble than sulfo-NHS esters.
  • the linker SMPT contains a sterically hindered disulfide bond, and can form antibody fusion protein with increased stability.
  • Disulfide linkages are in general, less stable than other linkages because the disulfide linkage is cleaved in vitro, resulting in less antibody fusion protein available.
  • Sulfo-NHS in particular, can enhance the stability of carbodimide couplings.
  • Carbodimide couplings (such as EDC) when used in conjunction with sulfo-NHS, forms esters that are more resistant to hydrolysis than the carbodimide coupling reaction alone.
  • compositions and methods for preparing the engineered cells described herein are also provided.
  • the binding moieties described herein comprise antibody moieties (for example anti-CCR5 antibody moiety, anti-HIV antibody moiety) .
  • the antibody moiety comprises V H and V L domains, or variants thereof, from the monoclonal antibody.
  • the antibody moiety further comprises C H 1 and C L domains, or variants thereof, from the monoclonal antibody.
  • Monoclonal antibodies can be prepared, e.g., using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256: 495 (1975) and Sergeeva et al., Blood, 117 (16) : 4262-4272.
  • a hamster, mouse, or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes can be immunized in vitro.
  • the immunizing agent can include a polypeptide or a fusion protein of the protein of interest, or a complex comprising at least two molecules, such as a complex comprising a peptide and an MHC protein.
  • PBLs peripheral blood lymphocytes
  • spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell.
  • a suitable fusing agent such as polyethylene glycol
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. Usually, rat or mouse myeloma cell lines are employed.
  • the hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ( “HAT medium” ) , which prevents the growth of HGPRT-deficient cells.
  • the immortalized cell lines fuse efficiently, support stable high-level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
  • the immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies. Kozbor, J. Immunol., 133: 3001 (1984) ; Brön et al. Monoclonal Antibody Production Techniques and Applications (Marcel Dekker, Inc.: New York, 1987) pp. 51-63.
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the polypeptide.
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells can be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA) . Such techniques and assays are known in the art.
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107: 220 (1980) .
  • the clones can be sub-cloned by limiting dilution procedures and grown by standard methods. Goding, supra. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by the sub-clones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the antibody moiety comprises sequences from a clone selected from an antibody moiety library (such as a phage library presenting scFv or Fab fragments) .
  • the clone may be identified by screening combinatorial libraries for antibody fragments with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics.
  • repertoires of V H and V L genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994) .
  • Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments.
  • naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self-antigens without any immunization as described by Griffiths et al., EMBO J, 12: 725-734 (1993) .
  • naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992) .
  • Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373, and US Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.
  • the antibody moiety can be prepared using phage display to screen libraries for antibodies specific to the target antigen (such as a CCR5 polypeptides) .
  • the library can be a human scFv phage display library having a diversity of at least one x 10 9 (such as at least about any of 1 x 10 9 , 2.5 x 10 9 , 5 x 10 9 , 7.5 x 10 9 , 1 x 10 10 , 2.5 x 10 10 , 5 x 10 10 , 7.5 x 10 10 , or 1 x 10 11 ) unique human antibody fragments.
  • the library is a human library constructed from DNA extracted from human PMBCs and spleens from healthy donors, encompassing all human heavy and light chain subfamilies.
  • the library is a human library constructed from DNA extracted from PBMCs isolated from patients with various diseases, such as patients with autoimmune diseases, cancer patients, and patients with infectious diseases.
  • the library is a semi-synthetic human library, wherein heavy chain CDR3 is completely randomized, with all amino acids (with the exception of cysteine) equally likely to be present at any given position (see, e.g., Hoet, R.M. et al., Nat. Biotechnol. 23 (3) : 344-348, 2005) .
  • the heavy chain CDR3 of the semi-synthetic human library has a length from about 5 to about 24 (such as about any of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24) amino acids.
  • the library is a fully synthetic phage display library.
  • the library is a non-human phage display library.
  • Phage clones that bind to the target antigen with high affinity can be selected by iterative binding of phage to the target antigen, which is bound to a solid support (such as, for example, beads for solution panning or mammalian cells for cell panning) , followed by removal of non-bound phage and by elution of specifically bound phage.
  • a solid support such as, for example, beads for solution panning or mammalian cells for cell panning
  • the target antigen can be biotinylated for immobilization to a solid support.
  • the biotinylated target antigen is mixed with the phage library and a solid support, such as streptavidin-conjugated Dynabeads M-280, and then target antigen-phage-bead complexes are isolated.
  • the bound phage clones are then eluted and used to infect an appropriate host cell, such as E. coli XL1-Blue, for expression and purification.
  • an appropriate host cell such as E. coli XL1-Blue
  • cells expressing the target antigen e.g., CCR5
  • the panning can be performed for multiple (such as about any of 2, 3, 4, 5, 6 or more) rounds with either solution panning, cell panning, or a combination of both, to enrich for phage clones binding specifically to the target antigen.
  • Enriched phage clones can be tested for specific binding to the target antigen by any methods known in the art, including for example ELISA and FACS.
  • the binding moieties bind to the same epitope as a reference antibody. In some embodiments, the binding moieties compete for binding with a reference antibody.
  • Competition assays can be used to determine whether two antibodies moieties bind the same epitope (or compete with each other) by recognizing identical or sterically overlapping epitopes or one antibody competitively inhibits binding of another antibody to the antigen. Exemplary competition assays include, but are not limited to, routine assays such as those provided in Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. ) .
  • the antibody moieties described herein can be human or humanized.
  • Humanized forms of non-human (e.g., murine) antibody moieties are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab’, F (ab’) 2 , scFv, or other antigen-binding subsequences of antibodies) that typically contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibody moieties include human immunoglobulins, immunoglobulin chains, or fragments thereof (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibody moieties can also comprise residues that are found neither in the recipient antibody moiety nor in the imported CDR or framework sequences.
  • the humanized antibody moiety can comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • CDR regions correspond to those of a non-human immunoglobulin
  • FR regions are those of a human immunoglobulin consensus sequence.
  • a humanized antibody moiety has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain.
  • humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature, 321: 522-525 (1986) ; Riechmann et al., Nature, 332: 323-327 (1988) ; Verhoeyen et al., Science, 239: 1534-1536 (1988) ) , by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody moiety.
  • humanized antibody moieties are antibody moieties (U.S. Patent No. 4,816,567) , wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibody moieties are typically human antibody moieties in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • human antibody moieties can be generated.
  • transgenic animals e.g., mice
  • JH antibody heavy-chain joining region
  • human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated.
  • Human antibodies may also be generated by in vitro activated B cells (see U.S. Patents 5,567,610 and 5,229,275) or by using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991) ; Marks et al., J. Mol. Biol., 222: 581 (1991) . The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies. Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147 (1) : 86-95 (1991) .
  • amino acid sequence variants of the antigen-binding domains are contemplated.
  • Amino acid sequence variants of an antigen-binding domain may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antigen-binding domain, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antigen-binding domain. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.
  • antigen-binding domain variants having one or more amino acid substitutions are provided.
  • Sites of interest for substitutional mutagenesis include the HVRs and FRs of antibody moieties.
  • Amino acid substitutions may be introduced into an antigen-binding domain of interest and the products screened for a desired activity, e.g., retained/improved antigen binding or decreased immunogenicity.
  • Amino acids may be grouped into different classes according to common side-chain properties:
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
  • An exemplary substitutional variant is an affinity matured antibody moiety, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques. Briefly, one or more CDR residues are mutated and the variant antibody moieties displayed on phage and screened for a particular biological activity (e.g., binding affinity) . Alterations (e.g., substitutions) may be made in HVRs, e.g., to improve antibody moiety affinity. Such alterations may be made in HVR “hotspots, ” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol.
  • variable genes chosen for maturation are introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis) .
  • a secondary library is then created. The library is then screened to identify any antibody moiety variants with the desired affinity.
  • Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. HC-CDR3 and LC-CDR3 in particular are often targeted.
  • substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody moiety to bind antigen.
  • conservative alterations e.g., conservative substitutions as provided herein
  • Such alterations may be outside of HVR "hotspots" or SDRs.
  • each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.
  • a useful method for identification of residues or regions of an antigen-binding domain that may be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244: 1081-1085.
  • a residue or group of target residues e.g., charged residues such as arg, asp, his, lys, and glu
  • a neutral or negatively charged amino acid e.g., alanine or polyalanine
  • a crystal structure of an antigen-antigen-binding domain complex can be determined to identify contact points between the antigen-binding domain and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.
  • Amino acid sequence insertions include amino-and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include an antigen-binding domain with an N-terminal methionyl residue.
  • Other insertional variants of the antigen-binding domain include the fusion to the N-or C-terminus of the antigen-binding domain to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antigen-binding domain.
  • nucleic acids or a set of nucleic acids encoding the surface molecules, binding moieties, membrane domains described herein, as well as vectors comprising the nucleic acid (s) .
  • the expression of the surface molecules, binding moieties, membrane domains can be achieved by inserting the nucleic acid (s) into an appropriate expression vector, such that the nucleic acid (s) is operably linked to 5’ and/or 3’ regulatory elements, including for example a promoter (e.g., a lymphocyte-specific promoter) and a 3’ untranslated region (UTR) .
  • the vectors can be suitable for replication and integration in host cells. Typical cloning and expression vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.
  • the nucleic acid (s) can be cloned into a number of types of vectors.
  • the nucleic acid can be cloned into a vector including, but not limited to, a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid.
  • Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • the expression vector may be provided to a cell in the form of a viral vector.
  • Viral vector technology is well known in the art.
  • Viruses that are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers.
  • retroviruses provide a convenient platform for gene delivery systems.
  • a selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo.
  • retroviral systems are known in the art.
  • adenovirus vectors are used.
  • a number of adenovirus vectors are known in the art.
  • lentivirus vectors are used.
  • Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells.
  • Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.
  • promoter elements e.g., enhancers
  • promoters regulate the frequency of transcriptional initiation.
  • these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.
  • tk thymidine kinase
  • the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto.
  • CMV immediate early cytomegalovirus
  • EF-1 ⁇ Elongation Growth Factor-1 ⁇
  • constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) , human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatinine kinase promoter.
  • SV40 simian virus 40
  • MMTV mouse mammary tumor virus
  • HSV human immunodeficiency virus
  • LTR long terminal repeat
  • MoMuLV promoter MoMuLV promoter
  • an avian leukemia virus promoter an Epstein-Barr virus immediate early promoter
  • Rous sarcoma virus promoter as well as human gene promoter
  • the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors.
  • the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells.
  • Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.
  • Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences.
  • a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.
  • Suitable reporter genes may include genes encoding luciferase, ⁇ -galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene. Suitable expression systems are well known and may be prepared using known techniques or obtained commercially.
  • the construct with the minimal 5′ flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.
  • Exemplary methods to confirm the presence of the nucleic acid (s) in the mammalian cell include, for example, molecular biological assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; biochemical assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological methods (such as ELISAs and Western blots) .
  • molecular biological assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR
  • biochemical assays such as detecting the presence or absence of a particular peptide, e.g., by immunological methods (such as ELISAs and Western blots) .
  • Vectors may be selected, for example, from the group consisting of mammalian expression vectors and viral vectors (such as those derived from retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses) .
  • mammalian expression vectors such as those derived from retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • the nucleic acid encoding the surface molecule comprises a first nucleic acid sequence encoding the first binding moiety that specifically binds to a T cell surface antigen (e.g., CCR5) , asecond nucleic acid sequence encoding the second binding moiety that specifically binds to an HIV antigen, and a third nucleic acid sequence encoding the membrane domain (e.g., SEQ ID NO: 39) .
  • the surface molecule is encoded by one nucleic acid and expressed from one expression cassette of an expression vector.
  • the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art.
  • the expression vector can be transferred into a host cell by physical, chemical, or biological means.
  • Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. In some embodiments, the introduction of a polynucleotide into a host cell is carried out by calcium phosphate transfection.
  • Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.
  • Viral vectors, and especially retroviral vectors have become the most widely used method for inserting genes into mammalian, e.g., human, cells.
  • Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus 1, adenoviruses and adeno-associated viruses, and the like.
  • Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle) .
  • an exemplary delivery vehicle is a liposome.
  • lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo) .
  • the nucleic acid may be associated with a lipid.
  • the nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.
  • Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution.
  • Lipids are fatty substances that may be naturally occurring or synthetic lipids.
  • lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
  • assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.
  • molecular biological assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR
  • biochemical assays such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.
  • nucleic acids described herein may be transiently or stably incorporated in a cell (such as a stem cell, such as an immune cell, such as a T cell) .
  • the nucleic acid is transiently expressed in the engineered cell.
  • the nucleic acid may be present in the nucleus of the engineered cell in an extrachromosomal array comprising the heterologous gene expression cassette.
  • Nucleic acids may be introduced into the engineered mammalian using any transfection or transduction methods known in the art, including viral or non-viral methods.
  • non-viral transfection methods include, but are not limited to, chemical-based transfection, such as using calcium phosphate, dendrimers, liposomes, or cationic polymers (e.g., DEAE-dextran or polyethylenimine) ; non-chemical methods, such as electroporation, cell squeezing, sonoporation, optical transfection, impalefection, protoplast fusion, hydrodynamic delivery, or transposons; particle-based methods, such as using a gene gun, magnectofection or magnet assisted transfection, particle bombardment; and hybrid methods, such as nucleofection.
  • the nucleic acid is a DNA.
  • the nucleic acid is a RNA.
  • the nucleic acid is linear.
  • the nucleic acid is circular.
  • the nucleic acid (s) is present in the genome of the engineered cell.
  • the nucleic acid (s) may be integrated into the genome of the cell by any methods known in the art, including, but not limited to, virus-mediated integration, random integration, homologous recombination methods, and site-directed integration methods, such as using site-specific recombinase or integrase, transposase, Transcription activator-like effector nuclease CRISPR/Cas9, and zinc-finger nucleases.
  • the nucleic acid (s) is integrated in a specifically designed locus of the genome of the engineered cell.
  • the nucleic acid (s) is integrated in an integration hotspot of the genome of the engineered cell. In some embodiments, the nucleic acid (s) is integrated in a random locus of the genome of the engineered cell. In the cases that multiple copies of the nucleic acids are present in a single engineered cell, the nucleic acid (s) may be integrated in a plurality of loci of the genome of the engineered cell.
  • the nucleic acid (s) encoding the surface molecule (s) can be operably linked to a promoter.
  • the promoter is an endogenous promoter.
  • the nucleic acid (s) encoding the surface molecule (s) may be knocked-in to the genome of the engineered cell downstream of an endogenous promoter using any methods known in the art, such as CRISPR/Cas9 method.
  • the endogenous promoter is a promoter for an abundant protein, such as beta-actin, CMV, or EF1 ⁇ .
  • the endogenous promoter is an inducible promoter, for example, inducible by an endogenous activation signal of the engineered cell.
  • the promoter is a T cell activation-dependent promoter (such as an IL-2 promoter, an NFAT promoter, or an NF ⁇ B promoter) .
  • the promoter is an autologous promoter.
  • the promoter is a heterologous promoter.
  • nucleic acid (s) encoding the surface molecule (s) is operably linked to a constitutive promoter. In some embodiments, the nucleic acid (s) encoding the surface molecule is operably linked to an inducible promoter.
  • a first promoter e.g., an inducible promoter
  • a second consecutive promoter e.g., a consecutive promoter
  • a T cell surface antigen e.g., CCR5
  • Constitutive promoters allow heterologous genes (also referred to as transgenes) to be expressed constitutively in the host cells.
  • Exemplary constitutive promoters contemplated herein include, but are not limited to, Cytomegalovirus (CMV) promoters, human elongation factors-1alpha (hEF1 ⁇ ) , ubiquitin C promoter (UbiC) , phosphoglycerokinase promoter (PGK) , simian virus 40 early promoter (SV40) , and chicken ⁇ -Actin promoter coupled with CMV early enhancer (CAGG) .
  • CMV Cytomegalovirus
  • hEF1 ⁇ human elongation factors-1alpha
  • UbiC ubiquitin C promoter
  • PGK phosphoglycerokinase promoter
  • SV40 simian virus 40 early promoter
  • CAGG chicken ⁇ -Actin promoter coupled with CMV early enhancer
  • the promoter in the nucleic acid is a hEF1 ⁇ promoter.
  • the inducible promoter can be induced by one or more conditions, such as a physical condition, microenvironment of the engineered cell, or the physiological state of the engineered cell, an inducer (i.e., an inducing agent) , or a combination thereof.
  • the inducing condition does not induce the expression of endogenous genes in the engineered cell, and/or in the subject that receives the pharmaceutical composition.
  • the inducing condition is selected from the group consisting of: inducer, irradiation (such as ionizing radiation, light) , temperature (such as heat) , redox state, tumor environment, and the activation state of the engineered cell.
  • the promoter is inducible by an inducer.
  • the inducer is a small molecule, such as a chemical compound.
  • the small molecule is selected from the group consisting of doxycycline, tetracycline, alcohol, metal, or steroids.
  • Chemically-induced promoters have been most widely explored. Such promoters includes promoters whose transcriptional activity is regulated by the presence or absence of a small molecule chemical, such as doxycycline, tetracycline, alcohol, steroids, metal and other compounds.
  • Doxycycline-inducible system with reverse tetracycline-controlled transactivator (rtTA) and tetracycline-responsive element promoter (TRE) is the most mature system at present.
  • WO9429442 describes the tight control of gene expression in eukaryotic cells by tetracycline responsive promoters.
  • WO9601313 discloses tetracycline-regulated transcriptional modulators.
  • Tet technology such as the Tet-on system, has described, for example, on the website of TetSystems. com. Any of the known chemically regulated promoters may be used to drive expression of the therapeutic protein in the present application.
  • the inducer is a polypeptide, such as a growth factor, a hormone, or a ligand to a cell surface receptor, for example, a polypeptide that specifically binds an HIV antigen.
  • the polypeptide is expressed by the engineered cell.
  • the polypeptide is encoded by a nucleic acid in the nucleic acid.
  • Many polypeptide inducers are also known in the art, and they may be suitable for use in the present invention. For example, ecdysone receptor-based gene switches, progesterone receptor-based gene switches, and estrogen receptor based gene switches belong to gene switches employing steroid receptor derived transactivators (WO9637609 and WO9738117 etc. ) .
  • the inducer comprises both a small molecule component and one or more polypeptides.
  • inducible promoters that dependent on dimerization of polypeptides are known in the art, and may be suitable for use in the present invention.
  • the first small molecule CID system developed in 1993, used FK1012, a derivative of the drug FK506, to induce homo-dimerization of FKBP.
  • Wu et al successfully make the CAR-T cells titratable through an ON-switch manner by using Rapalog/FKPB-FRB*and Gibberelline/GID1-GAI dimerization dependent gene switch (C. -Y.
  • dimerization dependent switch systems include Coumermycin/GyrB-GyrB (Nature 383 (6596) : 178-81) , and HaXS/Snap-tag-HaloTag (Chemistry and Biology 20 (4) : 549-57) .
  • the promoter is a light-inducible promoter, and the inducing condition is light.
  • Light inducible promoters for regulating gene expression in mammalian cells are also well known in the art (see, for example, Science 332, 1565-1568 (2011) ; Nat. Methods 9, 266-269 (2012) ; Nature 500: 472-476 (2013) ; Nature Neuroscience 18: 1202-1212 (2015) ) .
  • Such gene regulation systems can be roughly put into two categories based on their regulations of (1) DNA binding or (2) recruitment of a transcriptional activation domain to a DNA bound protein.
  • UVB ultraviolet B
  • the promoter is a light-inducible promoter that is induced by a combination of a light-inducible molecule, and light.
  • a light-cleavable photocaged group on a chemical inducer keeps the inducer inactive, unless the photocaged group is removed through irradiation or by other means.
  • Such light-inducible molecules include small molecule compounds, oligonucleotides, and proteins.
  • caged ecdysone, caged IPTG for use with the lac operon, caged toyocamycin for ribozyme-mediated gene expression, caged doxycycline for use with the Tet-on system, and caged Rapalog for light mediated FKBP/FRB dimerization have been developed (see, for example, Curr Opin Chem Biol. 16 (3-4) : 292-299 (2012) ) .
  • the promoter is a radiation-inducible promoter
  • the inducing condition is radiation, such as ionizing radiation.
  • Radiation inducible promoters are also known in the art to control transgene expression. Alteration of gene expression occurs upon irradiation of cells.
  • a group of genes known as “immediate early genes” can react promptly upon ionizing radiation.
  • exemplary immediate early genes include, but are not limited to, Erg-1, p21/WAF-1, GADD45alpha, t-PA, c-Fos, c-Jun, NF-kappaB, and AP1.
  • the immediate early genes comprise radiation responsive sequences in their promoter regions.
  • Consensus sequences CC (A/T) 6 GG have been found in the Erg-1 promoter, and are referred to as serum response elements or known as CArG elements.
  • Combinations of radiation induced promoters and transgenes have been intensively studied and proven to be efficient with therapeutic benefits. See, for example, Cancer Biol Ther. 6 (7) : 1005-12 (2007) and Chapter 25 of Gene and Cell Therapy: Therapeutic Mechanisms and Strategies, Fourth Edition CRC Press, Jan. 20 th , 2015. Any of the immediate early gene promoters or any promoter comprising a serum response element may be useful as a radiation inducible promoter to drive the expression of the therapeutic protein of the present invention.
  • the promoter is a heat inducible promoter, and the inducing condition is heat.
  • Heat inducible promoters driving transgene expression have also been widely studied in the art.
  • Heat shock or stress protein (HSP) including Hsp90, Hsp70, Hsp60, Hsp40, Hsp10 etc. plays important roles in protecting cells under heat or other physical and chemical stresses.
  • HSP heat shock or stress protein
  • GADD growth arrest and DNA damage
  • the promoter is inducible by a redox state.
  • exemplary promoters that are inducible by redox state include inducible promoter and hypoxia inducible promoters.
  • HIF-2 ⁇ restricts HIV transcription via direct binding to the viral promoter.
  • Hypoxia reduced tumor necrosis factor or histone deacetylase inhibitor, Romidepsin mediated reactivation of HIV and inhibiting HIF signaling-pathways reversed this phenotype. (See Zhuang et al., Commun Biol 3, 376 (2020) . ) .
  • the promoter is inducible by the physiological state, such as an endogenous activation signal, of the engineered cell.
  • the engineered cell is a stem cell (e.g., hematopoietic stem cell)
  • the promoter is a stem cell (e.g., hematopoietic stem cell) activation-dependent promoter.
  • the engineered cell is a T cell
  • the promoter is a T cell activation-dependent promoter, which is inducible by the endogenous activation signal of the engineered T cell.
  • the engineered T cell is activated by an inducer, such as PMA, ionomycin, or phytohaemagglutinin.
  • the engineered T cell is activated by recognition of HIV antigen via an endogenous T cell receptor, or an engineered receptor (such as recombinant TCR, or CAR) .
  • the engineered T cell is activated by blockade of an immune checkpoint, such as by an immunomodulator expressed by the engineered T cell or by a second engineered cell.
  • the T cell activation-dependent promoter is an IL-2 promoter.
  • the T cell activation-dependent promoter is an NFAT promoter.
  • the T cell activation-dependent promoter is a NF ⁇ B promoter.
  • IL-2 expression initiated by the gene transcription from IL-2 promoter is a major activity of T cell activation.
  • PMA Phorbol 12-myristate 13-acetate
  • ionomycin Phorbol 12-myristate 13-acetate
  • phytohaemagglutinin results in IL-2 secretion from stimulated T cells.
  • IL-2 promoter was explored for activation-induced transgene expression in genetically engineered T-cells (Virology Journal 3: 97 (2006) ) .
  • IL-2 promoter is efficient to initiate reporter gene expression in the presence of PMA/PHA-P activation in human T cell lines.
  • NFAT Nuclear Factor of Activated T cells
  • IL-2 interleukine-2
  • NFAT promoter is efficient to initiate reporter gene expression in the presence of PMA/PHA-P activation in human T cell lines.
  • Other pathways including nuclear factor kappa B (NF ⁇ B) can also be employed to control transgene expression via T cell activation.
  • the engineered cells are stem cells.
  • the stem cells are hematopoietic stem cells (e.g., CD34+ and/or CD33-cells) .
  • the stem cells may be derived from placental cells, embryonic stem cells, induced pluripotent stem cells, or hematopoietic stem cells.
  • the hematopoietic stem cells can be obtained from bone marrow cells or peripheral blood mononuclear cells (PBMCs) .
  • PBMCs peripheral blood mononuclear cells
  • the engineered cells are immune cells.
  • the engineered immune cells may be obtained from peripheral blood, cord blood, bone marrow, tumor infiltrating lymphocytes, lymph node tissue, or thymus tissue.
  • the host cells may include placental cells, embryonic stem cells, induced pluripotent stem cells, or hematopoietic stem cells.
  • the cells may be obtained from humans, monkeys, chimpanzees, dogs, cats, mice, rats, and transgenic species thereof.
  • the cells may be obtained from established cell lines.
  • the engineered immune cell is derived from a stem cell.
  • the stem cell is an embryonic stem cell (ESC) .
  • the stem cell is hematopoietic stem cell (HSC) .
  • the stem cell is a mesenchymal stem cell.
  • the stem cell is an induced pluripotent stem cell (iPSC) .
  • the engineered cells expressing the surface molecule can be generated by introducing one or more nucleic acids (including for example a lentiviral vector) encoding the surface molecule into the cell.
  • the vector is a viral vector.
  • viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, lentiviral vector, retroviral vectors, vaccinia vector, herpes simplex viral vector, and derivatives thereof.
  • Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) , and in other virology and molecular biology manuals.
  • retroviruses provide a convenient platform for gene delivery systems.
  • the nucleic acid can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to the engineered cell in vitro or ex vivo.
  • retroviral systems are known in the art.
  • adenovirus vectors are used.
  • a number of adenovirus vectors are known in the art.
  • lentivirus vectors are used.
  • self-inactivating lentiviral vectors are used.
  • self-inactivating lentiviral vectors carrying the nucleic acid sequence (s) encoding the surface molecule can be packaged with protocols known in the art.
  • the resulting lentiviral vectors can be used to transduce a mammalian cell (such as primary human T cells) using methods known in the art.
  • the transduced or transfected mammalian cell is propagated ex vivo after introduction of the nucleic acid.
  • the transduced or transfected mammalian cell is cultured to propagate for at least about any of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, or 14 days.
  • the transduced or transfected mammalian cell is cultured for no more than about any of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, or 14 days.
  • the transduced or transfected mammalian cell is further evaluated or screened to select the engineered cell.
  • the introduction of one or more nucleic acids into the cell can be accomplished using techniques known in the art.
  • the engineered cells such as engineered stem cell or T cells
  • the engineered cells are able to self-renew, expand and/or differentiate in vivo, resulting in long-term persistence that can lead to sustained control of a disease.
  • a source of the cells is obtained from a subject.
  • the cells e.g., stem cells or immune cells
  • the cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • any number of cell lines available in the art may be used.
  • cells can be obtained from a unit of blood or bone marrow collected from a subject using any number of techniques known to the skilled artisan, such as FICOLL TM separation.
  • cells from the circulating blood or bone marrow of an individual are obtained by apheresis.
  • Stem cells can be obtained from an adult or neonatal umbilical cord whole leukocyte sample or whole bone marrow cell sample.
  • the hematopoietic stem cells can be derived from a source selected from the group consisting of bone marrow, peripheral blood, and neonatal umbilical cord blood.
  • the stem cells are human hematopoietic stem cells selected based upon one or more of the following markers: CD34+, CD59+, CD90/Thy1+, CD38low/-, c-Kit-/low, and Lin-.
  • the stem cells are mouse hematopoietic stem cells based upon one or more of the following markers: CD34low/-, SCA-1+, CD90/Thy1+/low, CD38+, c-Kit+, and Lin-.
  • Immune cells can be derived from apheresis products.
  • the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps.
  • the cells are washed with phosphate buffered saline (PBS) .
  • PBS phosphate buffered saline
  • the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations.
  • a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer's instructions.
  • a semi-automated “flow-through” centrifuge for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5
  • the cells may be resuspended in a variety of biocompatible buffers, such as Ca 2+ -free, Mg 2+ -free PBS, PlasmaLyte A, or other saline solutions with or without buffer.
  • the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.
  • immune cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL TM gradient or by counterflow centrifugal elutriation.
  • a specific subpopulation of T cells such as CD3 + , CD28 + , CD4 + , CD8 + , CD45RA + , and CD45RO + T cells, can be further isolated by positive or negative selection techniques.
  • T cells are isolated by incubation with anti-CD3/anti-CD28 (i.e., 3 ⁇ 28) -conjugated beads, such as M-450 CD3/CD28 T, for a time period sufficient for positive selection of the desired T cells.
  • the time period is about 30 minutes. In some embodiments, the time period ranges from 30 minutes to 36 hours or longer (including all ranges between these values) . In some embodiments, the time period is at least one, 2, 3, 4, 5, or 6 hours. In some embodiments, the time period is 10 to 24 hours. In some embodiments, the incubation time period is 24 hours. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types.
  • T cells can be preferentially selected for or against at culture initiation or at other time points during the process.
  • subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points.
  • multiple rounds of selection can also be used in the context of this invention. In some embodiments, it may be desirable to perform the selection procedure and use the “unselected” cells in the activation and expansion process. “Unselected” cells can also be subjected to further rounds of selection.
  • Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells.
  • One method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected.
  • a monoclonal antibody cocktail typically includes antibodies to CD 14, CD20, CD11b, CD 16, HLA-DR, and CD8.
  • it may be desirable to enrich for or positively select for regulatory T cells which typically express CD4 + , CD25 + , CD62Lhi, GITR + , and FoxP3 + .
  • T regulatory cells are depleted by anti-CD25 conjugated beads or other similar methods of selection.
  • the concentration of cells and surface can be varied. In some embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells) , to ensure maximum contact of cells and beads. For example, in some embodiments, a concentration of about 2 billion cells/ml is used. In some embodiments, a concentration of about 1 billion cells/ml is used. In some embodiments, greater than about 100 million cells/ml is used. In some embodiments, a concentration of cells of about any of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used.
  • a concentration of cells of about any of 75, 80, 85, 90, 95, or 100 million cells/ml is used. In some embodiments, a concentration of about 125 or about 150 million cells/ml is used.
  • Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells, or from samples where there are many tumor cells present (i.e., leukemic blood, tumor tissue, etc. ) . Such populations of cells may have therapeutic value and would be desirable to obtain. For example, using high concentration of cells allows more efficient selection of CD8 + T cells that normally have weaker CD28 expression.
  • the immune cells are expanded by contacting with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells.
  • T cell populations may be stimulated, such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore.
  • a ligand that binds the accessory molecule is used for co-stimulation of an accessory molecule on the surface of the T cells.
  • a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells.
  • an anti-CD3 antibody and an anti-CD28 antibody can be used as can other methods commonly known in the art (Berg et al., Transplant Proc. 30 (8) : 3975-3977, 1998; Haanen et al., J. Exp. Med. 190 (9) : 13191328, 1999; Garland et al., J. Immunol. Meth. 227 (1-2) : 53-63, 1999) .
  • the cells can be activated and expanded.
  • the engineered cell is modified to block or decrease the expression of CCR5.
  • Modifications of cells to disrupt gene expression include any such techniques known in the art, including for example RNA interference (e.g., siRNA, shRNA, miRNA) , gene editing (e.g., CRISPR-or TALEN-based gene knockout) , and the like.
  • engineered cells with reduced expression of CCR5 are generated using the CRISPR/Cas system.
  • CRISPR/Cas system of gene editing see for example Jian W &Marraffini LA, Annu. Rev. Microbiol. 69, 2015; Hsu PD et al., Cell, 157 (6) : 1262-1278, 2014; and O’Connell MR et al., Nature 516: 263–266, 2014.
  • Engineered T cells with reduced expression of one or both of the endogenous TCR chains of the T cell are generated, for example using TALEN-based genome editing.
  • the engineered cells, in particular allogeneic immune cells obtained from donors can be modified to inactivate components of TCR involved in MHC recognition. In some embodiments, the modified immune cells do not cause graft versus host disease.
  • the CCR5 gene (or TCR gene) is inactivated using CRISPR/Cas9 gene editing.
  • CRISPR/Cas9 involves two main features: a short guide RNA (gRNA) and a CRISPR-associated endonuclease or Cas protein.
  • the Cas protein is able to bind to the gRNA, which contains an engineered spacer that allows for directed targeting to, and subsequent knockout of, a gene of interest. Once targeted, the Cas protein cleaves the DNA target sequence, resulting in the knockout of the gene.
  • the CCR5 gene (or TCR gene) is inactivated using transcription activator-like effector nuclease -based genome editing.
  • -based genome editing involves the use of restriction enzymes that can be engineered for targeting to particular regions of DNA.
  • a transcription activator-like effector (TALE) DNA-binding domain is fused to a DNA cleavage domain.
  • TALE transcription activator-like effector
  • the TALE is responsible for targeting the nuclease to the sequence of interest, and the cleavage domain (nuclease) is responsible for cleaving the DNA, resulting in the removal of that segment of DNA and subsequent knockout of the gene.
  • the CCR5 gene (or TCR gene) is inactivated using zinc finger nuclease (ZFN) genome editing methods.
  • Zinc finger nucleases are artificial restriction enzymes that are comprised of a zinc finger DNA-binding domain and a DNA-cleavage domain.
  • ZFN DNA-binding domains can be engineered for targeting to particular regions of DNA.
  • the DNA-cleavage domain is responsible for cleaving the DNA sequence of interest, resulting in the removal of that segment of DNA and subsequent knockout of the gene.
  • RNA interference such as small interference RNA (siRNA) , microRNA, and short hairpin RNA (shRNA) .
  • siRNA molecules are 20-25 nucleotide long oligonucleotide duplexes that are complementary to messenger RNA (mRNA) transcripts from genes of interest.
  • mRNA messenger RNA
  • siRNAs target these mRNAs for destruction. Through targeting, siRNAs prevent mRNA transcripts from being translated, thereby preventing the protein from being produced by the cell.
  • the expression of the CCR5 gene is reduced by using anti-sense oligonucleotides.
  • Antisense oligonucleotides targeting mRNA are generally known in the art and used routinely for downregulating gene expressions. See Watts, J. and Corey, D (2012) J. Pathol. 226 (2) : 365-379. )
  • a method of enriching a heterogeneous cell population for engineered cells expressing a surface molecule according to any of the engineered cells described herein.
  • a method of enriching a population for engineered cells comprising a nucleic acid encoding a surface molecule that comprises a) a binding moiety (such as any of the binding moieties described herein) and b) a membrane domain, wherein the method comprises enrichment based upon the binding moiety.
  • the binding moiety specifically binds to CCR5.
  • a method of enriching a population for engineered cells comprising a nucleic acid encoding a bispecific surface molecule that comprises a) a first binding moiety (e.g., specific for CCR5) , b) a second binding moiety (e.g., specific for an HIV antigen) and c) a membrane domain, wherein the method comprises enrichment based upon any of the binding moieties.
  • a first binding moiety e.g., specific for CCR5
  • a second binding moiety e.g., specific for an HIV antigen
  • the method comprises incubating an agent that specifically binds to the binding moiety (or any one of the binding moieties) with the population of the engineered cells. In some embodiments, the method comprises incubating an agent that specifically binds to the membrane domain. In some embodiments, the agent is an antibody. In some embodiments, at least about 90%, 95%, 96%. 97%, 98%, or 99%purity of cells is achieved via enrichment method described herein.
  • a population of engineered cells produced according to the methods described herein can be enriched for by positive selection techniques based upon, e.g., the expression of the surface molecule (s) .
  • engineered cells such as engineered stem cells or T cells
  • the time period is about 30 minutes.
  • the time period ranges from 30 minutes to 36 hours or longer (including all ranges between these values) .
  • the time period is at least one, 2, 3, 4, 5, or 6 hours.
  • the time period is 10 to 24 hours. In some embodiments, the incubation time period is 24 hours.
  • the incubation time period is 24 hours.
  • the concentration of cells and the beads can be varied. In some embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells) , to ensure maximum contact of cells and beads. For example, in some embodiments, a concentration of about 2 billion cells/ml is used. In some embodiments, a concentration of about 1 billion cells/ml is used. In some embodiments, greater than about 100 million cells/ml is used. In some embodiments, a concentration of cells of about any of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used.
  • a concentration of cells of about any of 75, 80, 85, 90, 95, or 100 million cells/ml is used. In some embodiments, a concentration of about 125 or about 150 million cells/ml is used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of engineered cells that may weakly express the surface molecule (s) .
  • enrichment results in minimal or substantially no exhaustion of the engineered cells (e.g., immune cells) .
  • enrichment results in fewer than about 50% (such as fewer than about any of 45, 40, 35, 30, 25, 20, 15, 10, or 5%) of the engineered cells becoming exhausted.
  • Immune cell exhaustion can be determined by any means known in the art, including any means described herein.
  • enrichment results in minimal or substantially no differentiation of the engineered cells (e.g., stem cells, e.g., immune cells) .
  • enrichment results in fewer than about 50% (such as fewer than about any of 45, 40, 35, 30, 25, 20, 15, 10, or 5%) of the engineered cells becoming differentiated.
  • Cell (e.g., stem cells, e.g., immune cells) differentiation can be determined by any methods known in the art, including any methods described herein.
  • enrichment results in minimal or substantially no internalization of surface molecule (s) on the engineered cells. For example, in some embodiments, enrichment results in less than about 50% (such as less than about any of 45, 40, 35, 30, 25, 20, 15, 10, or 5%) of the surface molecule (s) on the engineered cells becoming internalized. Internalization of the surface molecule (s) on engineered cells can be determined by any methods known in the art, including any methods described herein.
  • enrichment results in increased proliferation of the engineered cells.
  • enrichment results in an increase of at least about 10% (such as at least about any of 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000%or more) in the number of engineered cells following enrichment.
  • a method of enriching a heterogeneous cell population for engineered cells expressing a surface molecule comprising: a) contacting the heterogeneous cell population with a molecule comprising a target molecule (such as CCR5, an HIV antigen) or one or more epitopes contained therein to form complexes comprising the engineered cell bound to the molecule comprising the engineered cell bound to the molecule; and b) separating the complexes from the heterogeneous cell population, thereby generating a cell population enriched for the engineered cells.
  • the molecule is immobilized, individually, to a solid support.
  • the solid support is particulate (such as beads) .
  • the solid support is a surface (such as the bottom of a well) .
  • the molecule is labelled, individually, with a tag.
  • the tag is a fluorescent molecule, an affinity tag, or a magnetic tag.
  • the method further comprises eluting the engineered cells from the first and/or second molecules and recovering the eluate.
  • the engineered immune cells are enriched for CD4+ and/or CD8+ cells, for example through the use of negative enrichment, whereby cell mixtures are purified using two-step purification methods involving both physical (column) and magnetic (MACS magnetic beads) purification steps (Gunzer, M. et al. (2001) J. Immunol. Methods 258 (1-2) : 55-63) .
  • populations of cells can be enriched for CD4+ and/or CD8+ cells through the use of T cell enrichment columns specifically designed for the enrichment of CD4+ or CD8+ cells.
  • cell populations can be enriched for CD4+ cells through the use of commercially available kits.
  • the commercially available kit is the EASYSEP TM Human CD4+ T Cell Enrichment Kit (Stemcell Technologies) . In other embodiments, the commercially available kit is the MAGNISORT TM Mouse CD4+ T cell Enrichment Kit (Thermo Fisher Scientific) .
  • engineered cell compositions such as pharmaceutical compositions, also referred to herein as formulations
  • an engineered cell such as a stem cell, such as a T cell
  • an engineered cell composition comprising a homogeneous cell population of engineered cells (such as a stem cell, such as a T cell) of the same cell type, comprising one or more nucleic acids encoding same surface molecule (s) , and optionally expressing the same surface molecule (s) .
  • the engineered cell is a stem cell (e.g., hematopoietic stem cell) .
  • the engineered cell is a T cell.
  • the engineered cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer T cell, and a ⁇ T cell.
  • the engineered cell composition is a pharmaceutical composition.
  • an engineered cell composition comprising a heterogeneous cell population comprising a plurality of engineered cell populations comprising engineered cells of different cell types, which comprise different nucleic acids encoding different surface molecules, and optionally expressing different surface molecules.
  • the pharmaceutical composition is suitable for administration to an individual, such as a human individual.
  • the pharmaceutical composition is suitable for injection.
  • the pharmaceutical composition is suitable for infusion.
  • the pharmaceutical composition is substantially free of cell culture medium.
  • the pharmaceutical composition is substantially free of endotoxins or allergenic proteins.
  • “substantially free” is less than about any of 10%, 5%, 1%, 0.1%, 0.01%, 0.001%, 1ppm or less of total volume or weight of the pharmaceutical composition.
  • the pharmaceutical composition is free of mycoplasma, microbial agents, and/or communicable disease agents.
  • the pharmaceutical composition of the present applicant may comprise any number of the engineered cells.
  • the pharmaceutical composition comprises a single copy of the engineered cell.
  • the pharmaceutical composition comprises at least about any of 1, 10, 100, 1000, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 or more copies of the engineered cells.
  • the pharmaceutical composition comprises a single type of engineered cell.
  • the pharmaceutical composition comprises at least two types of engineered cells, wherein the different types of engineered cells differ by their cell sources, cell types, expressed therapeutic proteins (e.g., surface molecule (s) ) , and/or promoters, etc.
  • cryopreserved/cryopreserving can be used interchangeably. Freezing includes freeze-drying.
  • cells can be harvested from a culture medium, and washed and concentrated into a carrier in a therapeutically effective amount.
  • exemplary carriers include saline, buffered saline, physiological saline, water, Hanks' solution, Ringer's solution, Nonnosol-R (Abbott Labs) , Plasma-Lyte A (R) (Baxter Laboratories, Inc., Morton Grove, IL) , glycerol, ethanol, and combinations thereof.
  • carriers can be supplemented with human serum albumin (HSA) or other human serum components or fetal bovine serum.
  • HSA human serum albumin
  • a carrier for infusion includes buffered saline with 5%HAS or dextrose.
  • Additional isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.
  • Carriers can include buffering agents, such as citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and/or trimethylamine salts.
  • buffering agents such as citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and/or trimethylamine salts.
  • Stabilizers refer to a broad category of excipients, which can range in function from a bulking agent to an additive, which helps to prevent cell adherence to container walls.
  • Typical stabilizers can include polyhydric sugar alcohols; amino acids, such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, and cyclitols, such as inositol; PEG; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycol
  • compositions can include a local anesthetic such as lidocaine to ease pain at a site of injection.
  • a local anesthetic such as lidocaine to ease pain at a site of injection.
  • Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol.
  • Therapeutically effective amounts of cells within compositions can be greater than 10 2 cells, greater than 10 3 cells, greater than 10 4 cells, greater than 10 5 cells, greater than 10 6 cells, greater than 10 7 cells, greater than 10 8 cells, greater than 10 9 cells, greater than 10 10 cells, or greater than 10 11 cells, including any values and ranges in between these values.
  • cells are generally in a volume of a liter or less, 500 ml or less, 250 ml or less or 100 ml or less.
  • density of administered cells is typically greater than 10 4 cells/ml, 10 7 cells/ml or 10 8 cells/ml.
  • nucleic acid compositions such as pharmaceutical compositions, also referred to herein as formulations
  • the nucleic acid composition is a pharmaceutical composition.
  • the nucleic acid composition further comprises any of an isotonizing agent, an excipient, a diluent, a thickener, a stabilizer, a buffer, and/or a preservative; and/or an aqueous vehicle, such as purified water, an aqueous sugar solution, a buffer solution, physiological saline, an aqueous polymer solution, or RNase free water.
  • the amounts of such additives and aqueous vehicles to be added can be suitably selected according to the form of use of the nucleic acid composition.
  • compositions and formulations disclosed herein can be prepared for administration by, for example, injection, infusion, perfusion, or lavage.
  • the compositions and formulations can further be formulated for bone marrow, intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, and/or subcutaneous injection.
  • the formulations to be used for in vivo administration must be sterile. This is readily accomplished by, e.g., filtration through sterile filtration membranes.
  • compositions of the present application are useful for therapeutic purposes.
  • the pharmaceutical compositions of the present application comprises a pharmaceutically acceptable excipient suitable for administration to an individual.
  • Suitable pharmaceutically acceptable excipient may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide) ; and preservatives.
  • the pharmaceutically acceptable excipient comprises autologous serum.
  • the pharmaceutically acceptable excipient comprises human serum.
  • the pharmaceutically acceptable excipient is non-toxic, biocompatible, non-immunogenic, biodegradable, and can avoid recognition by the host’s defense mechanism.
  • the excipient may also contain adjuvants such as preserving stabilizing, wetting, emulsifying agents and the like.
  • the pharmaceutically acceptable excipient enhances the stability of the engineered cell or the antibody or other therapeutic proteins secreted thereof.
  • the pharmaceutically acceptable excipient reduces aggregation of the antibody or other therapeutic proteins secreted by the engineered cell.
  • the final form may be sterile and may also be able to pass readily through an injection device such as a hollow needle. The proper viscosity may be achieved and maintained by the proper choice of excipients.
  • the pharmaceutical composition is formulated to have a pH in the range of about 4.5 to about 9.0, including for example pH ranges of about any one of 5.0 to about 8.0, about 6.5 to about 7.5, or about 6.5 to about 7.0.
  • the pharmaceutical composition can also be made to be isotonic with blood by the addition of a suitable tonicity modifier, such as glycerol.
  • the pharmaceutical composition is suitable for administration to a human. In some embodiments, the pharmaceutical composition is suitable for administration to a human by parenteral administration.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation compatible with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizing agents, and preservatives.
  • the formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a condition requiring only the addition of the sterile liquid excipient methods of treatment, methods of administration, and dosage regimens described herein (i.e., water) for injection, immediately prior to use.
  • the pharmaceutical composition is contained in a single-use vial, such as a single-use sealed vial.
  • the pharmaceutical composition is contained in a multi-use vial.
  • the pharmaceutical composition is contained in bulk in a container.
  • the pharmaceutical composition is cryopreserved.
  • the pharmaceutical composition is formulated for intravenous administration. In some embodiments, the pharmaceutical composition is formulated for subcutaneous administration. In some embodiments, the pharmaceutical composition is formulated for local administration to a tumor site. In some embodiments, the pharmaceutical composition is formulated for intratumoral injection.
  • the pharmaceutical composition must meet certain standards for administration to an individual.
  • the United States Food and Drug Administration has issued regulatory guidelines setting standards for cell-based immunotherapeutic products, including 21 CFR 610 and 21 CFR 610.13. Methods are known in the art to assess the appearance, identity, purity, safety, and/or potency of pharmaceutical compositions.
  • the pharmaceutical composition is substantially free of extraneous protein capable of producing allergenic effects, such as proteins of an animal source used in cell culture other than the engineered mammalian immune cells.
  • “substantially free” is less than about any of 10%, 5%, 1%, 0.1%, 0.01%, 0.001%, 1ppm or less of total volume or weight of the pharmaceutical composition.
  • the pharmaceutical composition is prepared in a GMP-level workshop. In some embodiments, the pharmaceutical composition comprises less than about 5 EU/kg body weight/hr of endotoxin for parenteral administration. In some embodiments, at least about 70%of the engineered cells in the pharmaceutical composition are alive for intravenous administration. In some embodiments, the pharmaceutical composition has a “no growth” result when assessed using a 14-day direct inoculation test method as described in the United States Pharmacopoeia (USP) .
  • USP United States Pharmacopoeia
  • a sample including both the engineered cells and the pharmaceutically acceptable excipient should be taken for sterility testing approximately about 48-72 hours prior to the final harvest (or coincident with the last re-feeding of the culture) .
  • the pharmaceutical composition is free of mycoplasma contamination.
  • the pharmaceutical composition is free of detectable microbial agents.
  • the pharmaceutical composition is free of communicable disease agents, such as HIV type I, HIV type II, HBV, HCV, Human T-lymphotropic virus, type I; and Human T-lymphotropic virus, type II.
  • the present application further provides methods of administering the engineered cells to treat an infectious disease, for example HIV.
  • the present application thus in some embodiments provides a method for treating an infectious disease in an individual comprising administering to the individual an effective amount of a composition (such as a pharmaceutical composition) comprising engineered cells according to any one of the embodiments described herein.
  • the viral infection is caused by a virus selected, for example, Human T cell leukemia virus (HTLV) and HIV (Human immunodeficiency virus) .
  • HTLV Human T cell leukemia virus
  • HIV Human immunodeficiency virus
  • HIV-1 is the cause of the global pandemic and is a virus with both high virulence and high infectivity. HIV-2, however, is prevalent only in West Africa and is neither as virulent nor as infectious as HIV-1. The differences in virulence and infectivity between HIV-1 and HIV-2 infections may be rooted in the stronger immune response mounted against viral proteins in HIV-2 infections leading to more efficient control in affected individuals (Leligdowicz, A. et al. (2007) J. Clin. Invest. 117 (10) : 3067-3074) . This may also be a controlling reason for the global spread of HIV-1 and the limited geographic prevalence of HIV-2.
  • the engineered cells are used for treating HIV-1 infections. In other embodiments, the engineered cells are used for treating HIV-2 infections. In some embodiments, the engineered cells are used for treating HIV-1 and HIV-2 infections.
  • a method of treating an individual infected with HIV comprising administering to the individual an effective amount of engineered stem cells (e.g., hematopoietic stem cells) comprising a) a first binding moiety that specifically binds to CCR5, b) a second binding moiety that specifically binds to an HIV antigen and prevents the binding of the HIV antigen to the engineered cell, and c) a membrane domain comprising a GPI attachment signal sequence (e.g., SEQ ID NO: 39) .
  • the surface molecule does not comprise an intracellular signaling domain.
  • the first binding moiety is a scFv comprising the amino acid sequence of SEQ ID NO: 1 or 2, or a variant comprising at least about 80%sequence identity.
  • the second binding moiety specifically binds to an HIV antigen and prevents the binding of the HIV antigen to the engineered cell.
  • the second binding moiety specifically binds to an HIV antigen competitively with 10E8, or binds to the same epitope as that of 10E8.
  • the second binding moiety is a scFv comprising the amino acid sequence of SEQ ID NO: 25, or a variant comprising at least about 80%sequence identity.
  • a method of treating an individual infected with HIV comprising administering to the individual an effective amount of engineered immune cells (e.g., T cells, NK cells) comprising a) a first binding moiety, wherein the first binding moiety specifically binds to CCR5, b) a second binding moiety, wherein the second binding moiety specifically binds to an HIV antigen, and c) a membrane domain comprising a GPI attachment signal sequence (e.g., SEQ ID NO: 39) .
  • the surface molecule does not comprise an intracellular signaling domain.
  • the first binding moiety is a scFv comprising the amino acid sequence of SEQ ID NO: 1 or 2, or a variant comprising at least about 80%sequence identity.
  • the second binding moiety specifically binds to an HIV antigen competitively with 10E8, or binds to the same epitope as that of 10E8.
  • the second binding moiety is a scFv comprising the amino acid sequence of SEQ ID NO: 25, or a variant comprising at least about 80%sequence identity.
  • the engineered cells are autologous to the individual.
  • the engineered cells are allogeneic to the individual.
  • At least about 5% (such as at least about 5%, 6%, 7%, 8%. 9%, 10%, 12%, 14%, 16%, 18%, or 20%) of the T cells in the individual express the surface molecule after administration of the engineered cells.
  • the T cell are measured about 1, 2, 3, 4, 5, 6, or 7 days after administration of the engineered cells.
  • the T cell are measured about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks after administration of the engineered cells.
  • the T cell are measured about 1, 2, 3, 4, 5, or 6 months after administration of the engineered cells.
  • At least about 20% (such as at least about 22%, 24%, 26%, 28%. 30%, 32%, 34%, 36%, 38%, or 40%) of the T cells in the individual are resistant to HIV infection after administration of the engineered cells.
  • the HIV resistance of the T cells are measured about 1, 2, 3, 4, 5, 6, or 7 days after administration of the engineered cells.
  • the HIV resistance of the T cells are measured about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks after administration of the engineered cells.
  • the HIV resistance of the T cells are measured about 1, 2, 3, 4, 5, or 6 months after administration of the engineered cells.
  • the individual is a mammal (e.g., human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc. ) .
  • the individual is a human.
  • the individual is a clinical patient, a clinical trial volunteer, an experimental animal, etc.
  • the individual is younger than about 60 years old (including for example younger than about any of 50, 40, 30, 25, 20, 15, or 10 years old) .
  • the individual is older than about 60 years old (including for example older than about any of 70, 80, 90, or 100 years old) .
  • the individual is diagnosed with or environmentally or genetically prone to one or more of the diseases or disorders described herein (such as cancer or viral infection) .
  • the individual has one or more risk factors associated with one or more diseases or disorders described herein.
  • the engineered cell compositions of the invention are administered in combination with a second, third, or fourth agent (including, e.g., an antineoplastic agent, a growth inhibitory agent, a cytotoxic agent, or a chemotherapeutic agent) to treat diseases or disorders involving target antigen expression.
  • a second, third, or fourth agent including, e.g., an antineoplastic agent, a growth inhibitory agent, a cytotoxic agent, or a chemotherapeutic agent
  • Viral infection treatments can be evaluated, for example, by viral load, duration of survival, quality of life, protein expression and/or activity.
  • the pharmaceutical composition is administered at a dosage of at least about any of 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 cells/kg of body weight. In some embodiments, the pharmaceutical composition is administered at a dosage of any of about 10 4 to about 10 5 , about 10 5 to about 10 6 , about 10 6 to about 10 7 , about 10 7 to about10 8 , about 10 8 to about 10 9 , about 10 4 to about 10 9 , about 10 4 to about 10 6 , about 10 6 to about 10 8 , or about 10 5 to about 10 7 cells/kg of body weight.
  • more than one type of engineered cells are administered, the different types of engineered cells may be administered to the individual simultaneously, such as in a single composition, or sequentially in any suitable order.
  • the pharmaceutical composition is administered for a single time. In some embodiments, the pharmaceutical composition is administered for multiple times (such as any of 2, 3, 4, 5, 6, or more times) . In some embodiments, the pharmaceutical composition is administered once per week, once 2 weeks, once 3 weeks, once 4 weeks, once per month, once per 2 months, once per 3 months, once per 4 months, once per 5 months, once per 6 months, once per 7 months, once per 8 months, once per 9 months, or once per year. In some embodiments, the interval between administrations is about any one of 1 week to 2 weeks, 2 weeks to 1 month, 2 weeks to 2 months, 1 month to 2 months, 1 month to 3 months, 3 months to 6 months, or 6 months to a year.
  • the optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.
  • the methods of treating an infectious disease described herein further comprises administering to the individual a second anti-infectious disease agent.
  • Suitable anti-infectious disease agents include, but are not limited to, anti-retroviral drugs, broad neutralization antibodies (bnAb) , toll-like receptor agonists, latency reactivation agents, CCR5 antagonists, immune stimulators (e.g., TLR ligands) , vaccines, nucleoside reverse transcriptase inhibitors, nucleotide reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, HIV protease inhibitors, and fusion inhibitors.
  • the second anti-infectious agent is administered simultaneously with the engineered cells.
  • the second anti-infectious agent is administered sequentially with (e.g., prior to or after) the administration of the engineered cells.
  • an article of manufacture containing materials useful for the treatment of an infectious disease such as viral infection (for example infection by HIV) .
  • the article of manufacture can comprise a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is effective for treating a disease or disorder described herein, and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle) .
  • At least one active agent in the composition is an engineered cell of the invention.
  • the label or package insert indicates that the composition is used for treating the particular condition.
  • the label or package insert will further comprise instructions for administering the engineered cell composition to the patient.
  • Articles of manufacture and kits comprising combinatorial therapies described herein are also contemplated.
  • Package insert refers to instructions customarily included in commercial packages of therapeutic products that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.
  • the package insert indicates that the composition is used for treating a target antigen-positive viral infection (for example infection by HIV) .
  • the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI) , phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI) , phosphate-buffered saline, Ringer's solution and dextrose solution.
  • Kits are also provided that are useful for various purposes, e.g., for treatment of a target antigen-positive disease or disorder described herein, optionally in combination with the articles of manufacture.
  • Kits of the invention include one or more containers comprising an engineered cell composition (or unit dosage form and/or article of manufacture) , and in some embodiments, further comprise another agent (such as the agents described herein) and/or instructions for use in accordance with any of the methods described herein.
  • the kit may further comprise a description of selection of individuals suitable for treatment.
  • Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit) , but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.
  • CD8 ⁇ signal peptide SEQ ID NO: 38
  • scFv -linker sequence -scFv -linker sequence
  • GPI attachment sequence any of SEQ ID NOs: 1-2, or 25
  • GPI-scFvs have from N-terminus to C-terminus: CD8 ⁇ signal peptide (SEQ ID NO: 38) - [scFv -linker sequence (-scFv -linker sequence) -GPI attachment sequence] (any of SEQ ID NOs: 1-2, or 25) -GFP (SEQ ID NO: 43)
  • GPI TZM-bl cells were produced as described below. TZM-bl cells were digested and centrifuged at 800 rpm for 5min. Cell pellet was resuspended and counted. 2E+05 cells were transferred to a 24-well plate and the cell growth medium was refilled to 1 mL. After 24 hours, the supernatant was discarded, and 1ml of complete medium containing lentivirus with GPI-scFvs constructs as listed in Table 3 was added to the cells for incubation. Cells were then digested, transferred into a 6-well plate and incubated. Green fluorescence under a fluorescence microscope was measured. GPI-559-TZM-bl cells were polyclonal and the purity was 62.4%.
  • the cells were then digested and re-plated in a 96-well plate using a serial dilution method for plating in 200 ⁇ l culture medium.
  • Cells were cultivated in the incubator continuously for 10 days.
  • An inverted microscope was used to observe whether there was a single cell cluster in the wells.
  • Single cell clusters were checked under a fluorescence microscope to confirm whether the clusters have green fluorescence.
  • Single cell clusters with green fluorescence were selected and expanded, and subjected to flow cytometry to detect the purity of monoclonal cells.
  • the results are shown in FIG. 1, where the monoclonal TZM-bl cell lines expressing surface GPI-scFVs or HIV bnAbs or GPI-bi-ScFVs have high purity, ranging from 97.9%to 99.7%.
  • Herd immunity generally refers to the resistance to the spread of a contagious disease within a population that results if a sufficiently high proportion of individuals are immune to the disease, especially through vaccination. We tested whether cell-level herd immunity can be achieved by mixing HIV-resistant cells with HIV-nonresistant cells.
  • Blank TZM-bl cells and GPI TZM-bl cells were mixed at 100%, 75%, 60%, 45%, 35%, 15%of GPI TZM-bl cells.
  • GPI TZM-bl cells used include mono-GPI-C1-13 (i.e., monoclonal GPI TMZ-bl cells expressing anti-CCR5 scFv, similar abbreviations for cells described below) , Poly-GPI-10E8, mono-GPI-604, mono-GPI-615. Culture medium was refilled to 200ul. Cells were then incubated with pseudovirus for 24 hours. Positive group (PC) was set to be blank TZM-bl cells with pseudovirus infection. 100%GPI TZM-bl cells without virus infection were set up as negative control (NC) groups. Another negative control group of blank TZM-bl cells without pseudovirus infection was also tested.
  • PC Positive group
  • NC negative control
  • GPI-anchored HIV-resistant protein e.g., antibody moieties specifically binding to CCR5, HIV antigen
  • X-axis the percentage of cells protected against infection (blocking effect) was denoted as Y-axis.
  • GPI anchored anti-CCR5 (GPI-C1-13) scFv expressing cells exhibited cell-level herd immunity, conferring more than 50%blocking at only 15%of the cell population, and approximately 90%protection at 50%population.
  • GPI-604 a single clone expressing GPI-C1-13-10E8, exhibited better herd immunity compared to single ScFv moiety, while GPI-615, a single clone expressing GPI-10E8-C1-13, has a gentle blocking effect.
  • GPI TZM-bl cells were produced as described below. TZM-bl cells were digested and centrifuged at 800 rpm for 5min. Cell pellet was resuspended and counted. 2E+05 cells were transferred to a 24-well plate and the cell growth medium was refilled to 1 mL. After 24 hours, the supernatant was discarded, and 1ml of complete medium containing lentivirus with GPI-scFvs constructs as described above was added to the cells for incubation. Cells were then digested, transferred into a 6-well plate and incubated. Green fluorescence under a fluorescence microscope was measured.
  • Single cell clusters with green fluorescence were selected and expanded, and subjected to flow cytometry to detect the purity of monoclonal cells. And selected the GPI-604 clone 2 with a purity of 98.7%and the GPI-604 clone 6 with a purity of 99.7%, as shown in FIG 3.
  • MFI of the scFv expression of GPI-604 clone 2 and clone 6 were 1575 and 25000, respectively.
  • GPI-604 cells As shown in FIG 4, the percentage of GPI-604 cells was denoted as X-axis and the percentage of cells protected against infection (blocking effect) was denoted as Y-axis.

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Abstract

L'invention concerne des cellules modifiées (telles que des cellules souches ou des cellules T) qui ont une molécule de surface comprenant une fraction de liaison attachée à une membrane qui se lie à un antigène de surface de cellule T (tel que CCR5) et un antigène du VIH. L'invention concerne également des procédés de fabrication et d'utilisation de ces cellules modifiées.
PCT/CN2023/106274 2022-07-08 2023-07-07 Cellules modifiées et leurs utilisations WO2024008177A1 (fr)

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