CXCR4 antibody-resistant modified receptor

Abstract

A method is provided for improving the engraftment of donor cells in a subject. The method may include the steps of: providing cells modified to express a first isoform of a target protein (e.g., CXC-motif chemokine receptor 4 (CXCR4)); administering the donor cells to a subject; and then selectively inhibiting host cells in the subject based on the expression of a second isoform of the target protein, thereby improving the engraftment of the donor cells in the subject. Also provided is a combination for administration to a subject in need, comprising (1) a population of donor cells modified to express a first isoform of the target protein (e.g., CXCR4); and (2) an antagonist (e.g., an anti-CXCR4 antigen-binding protein) that specifically binds to the second isoform of the target protein but not to the first isoform of the target protein.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the interests of U.S. Application No. 63 / 579,165 filed on 28 August 2023 and U.S. Application No. 63 / 611,395 filed on 18 December 2023, each of which is incorporated herein by reference in whole for all purposes.

[0002] References to sequence listings submitted as XML files The sequence listing contained in file 617845SEQLIST.xml is 140,705 bytes in size, was created on August 26, 2024, and is incorporated herein by reference. [Background technology]

[0003] background Hematopoietic stem cell transplantation (HSCT) can completely replace the hematopoietic system of a transplant recipient with donor-derived cells. This technique can be curative for hematological malignancies, genetic defects in bloodlines, and severe autoimmune diseases refractory to standard treatment. However, patients must first undergo a procedure called host adaptation, which partially or completely ablates their immune system to facilitate HSCT graft uptake. Traditional methods for transplant host adaptation use genotoxic substances such as chemotherapeutic agents and radioactive materials that have little specificity for the desired target cells. The associated risks of off-target tissue damage, secondary malignancies, and opportunistic infections thus limit the application of HSCT as a last resort.

[0004] Monoclonal antibodies (mAbs) and related therapies enable the targeting of specific cell populations, thus paving the way for safer and more effective conditioning regimens. Host hematopoietic stem cells (HSCs) can be depleted by antibodies that block the function of essential surface molecules or are involved in Fc-mediated effector mechanisms. Alternatively, agents that replace HSCs from their resident niche in the bone marrow (BM) may facilitate the engraftment of donor HSCs. Blockade of CXC-motif chemokine receptor 4 (CXCR4), a G protein-coupled receptor (GPCR) essential for the localization of hematopoietic cells to and within the BM, is known to induce maturation from the BM to peripheral sites and the recruitment of precursor immune cells. Small molecules, peptides, and mAbs that block the interaction between CXCR4 and its ligand, CXC-motif chemokine 12 (CXCL12, also known as SDF1), have this effect in preclinical models and patients. However, a major challenge with such conditioning methods is that donor HSCs, along with the host, are susceptible to CXCR4 blockers during the engraftment window. [Overview of the project] [Means for solving the problem]

[0005] overview Methods for improving donor cell engraftment in subjects requiring such improvement are provided. Combinations or combination pharmaceuticals for administration to subjects requiring such improvement are also provided. Isolated cells or populations of cells modified to express a first isoform of CXC chemokine receptor 4 (CXCR4) different from a second isoform of CXCR4 are also provided. Methods for producing isolated cells or populations of cells are also provided. Genetically modified CXC chemokine receptor 4 (CXCR4) protein and nucleic acids encoding the protein are also provided.

[0006] In one embodiment, a method is provided for improving the engraftment of donor cells in a subject that requires it. Some such methods include the steps of (a) providing donor cells that have been modified to express a first isoform of CXCR4 that is different from a second isoform of CXCR4, wherein the second isoform is expressed in host cells of the subject; (b) administering the donor cells to the subject; and (c) selectively inhibiting host cells in the subject based on the expression of the second isoform of CXCR4, thereby improving the engraftment of donor cells in the subject.

[0007] In some such methods, selective inhibition of host cells in step (c) does not involve ablation of host cells by an active killing mechanism. In some such methods, selective inhibition in step (c) involves selective depletion of host cells from the bone marrow. In some such methods, the first and second isoforms are functionally indistinguishable but immunologically distinguishable. In some such methods, donor cells express both the first and second isoforms of CXCR4. In some such methods, donor cells express only the first isoform of CXCR4. In some such methods, the first isoform of CXCR4 is expressed in donor cells from an expression vector, or a genomic locus is edited to express the first isoform of CXCR4 in donor cells. In some such methods, the genomic locus is the endogenous CXCR4 genomic locus. In some such methods, the genomic locus is not the endogenous CXCR4 genomic locus.

[0008] In some such methods, selective inhibition in step (c) involves administering a CXCR4 antagonist to a target, the CXCR4 antagonist specifically binding to a second isoform of CXCR4 but not specifically binding to a first isoform of CXCR4, and, if necessary, step (c) includes multiple administrations of the CXCR4 antagonist. In some such methods, the CXCR4 antagonist is an antigen-binding protein. In some such methods, the antigen-binding protein is an antibody or an antigen-binding fragment thereof. In some such methods, the antigen-binding protein comprises an immunoglobulin light chain or its variable region containing three light chain CDRs and an immunoglobulin heavy chain or its variable region containing three heavy chain CDRs, wherein each of the three light chain CDRs contains, essentially, or comprises a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 15, 17, and 19, and each of the three heavy chain CDRs contains, essentially, or comprises a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 7, 9, and 11. In some such methods, the antigen-binding protein includes an immunoglobulin light chain variable region containing, essentially, or consisting of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 13, and the antigen-binding protein includes an immunoglobulin heavy chain variable region containing, essentially, or consisting of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 5. In some such methods, the immunoglobulin light chain variable region includes, essentially, or consists of the sequence described in SEQ ID NO: 13, and the immunoglobulin heavy chain variable region includes, essentially, or consists of the sequence described in SEQ ID NO: 5.In some such methods, the antigen-binding protein comprises an immunoglobulin light chain or its variable region containing three light chain CDRs and an immunoglobulin heavy chain or its variable region containing three heavy chain CDRs, wherein each of the three light chain CDRs contains, essentially, or comprises a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 31, 33, and 35, and each of the three heavy chain CDRs contains, essentially, or comprises a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 23, 25, and 27. In some such methods, the antigen-binding protein includes an immunoglobulin light chain variable region containing, essentially, or consisting of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 29, and the antigen-binding protein includes an immunoglobulin heavy chain variable region containing, essentially, or consisting of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 21. In some such methods, the immunoglobulin light chain variable region includes, essentially, or consists of the sequence described in SEQ ID NO: 29, and the immunoglobulin heavy chain variable region includes, essentially, or consists of the sequence described in SEQ ID NO: 21.

[0009] In some such methods, the first isoform of CXCR4 is a genetically engineered isoform of CXCR4. In some such methods, the first isoform of CXCR4 is genetically engineered to contain a mutation that provides an altered epitope, and if necessary, the mutation is an artificial mutation. In some such methods, the altered epitope is located in the binding region of a CXCR4 antagonist, and as a result, the CXCR4 antagonist exhibits a reduced or lost ability to bind to and / or inhibit the first isoform of CXCR4 compared to its ability to bind to and / or inhibit the second isoform of CXCR4. In some such methods, both the first and second isoforms of CXCR4 retain the ability to bind to an endogenous ligand, and if necessary, the CXCR4 antagonist blocks the binding of the endogenous ligand to the second isoform of CXCR4 but not to the first isoform of CXCR4.

[0010] In some such methods, the mutation is located in the extracellular loop 2 (ECL2) region of CXCR4. In some such methods, the mutation involves an insertion, deletion, or substitution within the region of CXCR4 from S178 to R183 and / or within the region of R188 to L194. In some such methods, the mutation comprises one or more mutations selected from F189A, N192A, D193A, S178_E179insK, S178_E179insY, S178_E179insR, E179R, D181R, and D182R, and optionally the mutation comprises (1) F189A, N192A, and D193A, (2) S178_E179insK, (3) S178_E179insY, (4) S178_E179insR, or (5) E179R, D181R, and D182R.

[0011] In some such methods, the donor cells and / or host cells are hematopoietic cells, and, if necessary, the donor cells and / or host cells are immune cells. In some such methods, the donor cells and / or host cells are lymphocytes or lymphoid progenitor cells. In some such methods, the donor cells and / or host cells are T cells. In some such methods, the donor cells and / or host cells are alpha-beta T cells. In some such methods, the donor cells and / or host cells are gamma-delta T cells. In some such methods, the donor cells and / or host cells are tumor-infiltrating lymphocytes (TILs). In some such methods, the donor cells and / or host cells are B cells, and, if necessary, the donor cells and / or host cells are immature B cells, and this method depletes the host's mature B cells from the bone marrow. In some such methods, the donor cells and / or host cells are NK cells. In some such methods, the donor cells and / or host cells are hematopoietic stem and progenitor cells. In some such methods, the donor cells and / or host cells are derived from hematopoietic stem cells or hematopoietic stem and progenitor cells. In some such methods, the donor cells are derived from induced pluripotent stem cells. In some such methods, the subject is a mammal or a non-human mammal, and the donor cells are mammalian cells or non-human mammalian cells. In some such methods, the subject is a human, and the donor cells are human cells. In some such methods, the donor cells contain or express a therapeutic molecule. In some such methods, the therapeutic molecule does not target CXCR4. In some such methods, the donor cells contain or express an immunoglobulin, a chimeric antigen receptor (CAR), or an exogenous T cell receptor (TCR). In some such methods, the immunoglobulin, CAR, or exogenous TCR does not target CXCR4. In some such methods, the donor cells are autologous. In some such methods, donor cells are homogeneous or syngeneic.

[0012] In some such methods, the subject has a disease or disorder, and the method is for treating the disease or disorder in the subject. In some such methods, the subject has cancer. In some such methods, the cancer is a solid tumor carcinoma. In some such methods, the cancer is a hematological malignancy. In some such methods, the subject has a hematopoietic malignancy, and the method is for treating the hematopoietic malignancy in the subject. In some such methods, the subject has a deficient immune cell or hereditary hematopoietic failure. In some such methods, the hereditary hematopoietic failure is sickle cell disease or severe combined immunodeficiency (SCID).

[0013] In some such methods, steps (b) and (c) are performed simultaneously. In some such methods, step (b) is performed before step (c), and if necessary, step (c) includes multiple doses of the CXCR4 antagonist after step (b). In some such methods, step (b) is performed after step (c), and if necessary, step (c) includes multiple doses of the CXCR4 antagonist before step (b). In some such methods, step (c) is performed both before and after step (b), and if necessary, step (c) includes multiple doses of the CXCR4 antagonist before step (b) and / or multiple doses of the CXCR4 antagonist after step (b).

[0014] In some such methods, the method further includes a step of generating donor cells by modifying a population of cells to express a first isoform of CXCR4 prior to step (a). In some such methods, the population of cells is a population of induced pluripotent stem cells, and the method further includes a step of differentiating the induced pluripotent stem cells into donor cells to be administered in step (a) prior to step (a), and optionally differentiating the induced pluripotent stem cells into hematopoietic cells, lymphocytes or lymphoid progenitor cells, T cells, B cells, NK cells, hematopoietic stem cells, or hematopoietic stem and progenitor cells. In some such methods, the population of cells is a population of hematopoietic stem cells or hematopoietic stem and progenitor cells, and the method further includes a step of differentiating the hematopoietic stem cells or hematopoietic stem and progenitor cells into donor cells to be administered in step (a) prior to step (a), and optionally differentiating the hematopoietic stem cells or hematopoietic stem and progenitor cells into differentiated hematopoietic cells, lymphocytes or lymphoid progenitor cells, T cells, B cells, or NK cells. In some such methods, the step of generating donor cells includes, prior to step (a), introducing an expression vector encoding a first isoform of CXCR4 to express a first isoform of CXCR4, or the step of generating donor cells includes, prior to step (a), editing a genomic locus in a population of cells to express a first isoform of CXCR4. In some such methods, the genomic locus is an endogenous CXCR4 genomic locus. In some such methods, the genomic locus is not an endogenous CXCR4 genomic locus.In some such methods, the editing step includes introducing (1) a nuclease agent or one or more nucleic acids encoding a nuclease agent, wherein the nuclease agent targets a nuclease target sequence at a genomic locus, and (2) an exogenous donor nucleic acid into a population of cells, wherein the nuclease agent cleaves the genomic locus, and the exogenous donor nucleic acid is inserted into or recombined with the genomic locus to produce donor cells expressing a first isoform of CXCR4. In some such methods, the nuclease agent comprises (a) a zinc finger nuclease (ZFN), (b) a transcription activator-like effector nuclease (TALEN), or (c) a guide RNA comprising (i) a Cas protein and (ii) a DNA targeting segment that targets a guide RNA target sequence which is a nuclease target sequence, wherein the guide RNA binds to the Cas protein and directs the Cas protein to the guide RNA target sequence. In some such methods, the nuclease agent comprises a Cas protein and a guide RNA, and optionally the DNA targeting segment comprises a sequence described in any one of SEQ ID NOs: 132-146, or optionally the guide RNA target sequence comprises a sequence described in any one of SEQ ID NOs: 117-131. In some such methods, the Cas protein is a Cas9 protein. In some such methods, the exogenous donor nucleic acid comprises a homology arm. In some such methods, the exogenous donor nucleic acid is a single-stranded oligodeoxynucleotide (ssODN). In some such methods, the method further includes the step of isolating a population of cells from or from a different subject prior to the step of modifying the population of cells.

[0015] Some such methods include (a) providing donor cells modified to express a first isoform of CXCR4, wherein the first isoform of CXCR4 differs from a second isoform of CXCR4, the second isoform being expressed in host cells of interest; (b) administering the donor cells to an interest; and (c) providing the interest with means to specifically bind to the second isoform of CXCR4 but not to the first isoform of CXCR4.

[0016] In some such methods, means for specifically binding to a second isoform of CXCR4 but not to a first isoform of CXCR4 selectively inhibit host cells in a target based on the expression of the second isoform of CXCR4. In some such methods, selective inhibition of host cells does not involve ablation of host cells by an active killing mechanism. In some such methods, selective inhibition of host cells includes selective depletion of host cells from the bone marrow.

[0017] In some such methods, the first and second isoforms are functionally indistinguishable but immunologically distinguishable. In some such methods, donor cells express both the first and second isoforms of CXCR4. In some such methods, donor cells express only the first isoform of CXCR4. In some such methods, the first isoform of CXCR4 is expressed in donor cells from an expression vector, or a genomic locus is edited to express the first isoform of CXCR4 in donor cells. In some such methods, the genomic locus is the endogenous CXCR4 genomic locus. In some such methods, the genomic locus is not the endogenous CXCR4 genomic locus.

[0018] In some such methods, the first isoform of CXCR4 is a genetically engineered isoform of CXCR4. In some such methods, the first isoform of CXCR4 is genetically engineered to contain a mutation that provides an altered epitope, and if necessary, the mutation is an artificial mutation. In some such methods, the altered epitope is located in the binding region of a means to specifically bind to the second isoform of CXCR4 but not to the first isoform of CXCR4, and as a result, the means to specifically bind to the second isoform of CXCR4 but not to the first isoform of CXCR4 exhibits a reduction or loss of ability to bind to and / or inhibit the first isoform of CXCR4 compared to its ability to bind to and / or inhibit the second isoform of CXCR4. In some such methods, both the first and second isoforms of CXCR4 retain the ability to bind to the endogenous ligand, and, if necessary, specifically bind to the second isoform of CXCR4 but not specifically to the first isoform of CXCR4. The means to do this is to block the binding of the endogenous ligand to the second isoform of CXCR4 but not to the first isoform of CXCR4. In some such methods, the mutation is located in the extracellular loop 2 (ECL2) region of CXCR4. In some such methods, the mutation includes insertions, deletions, or substitutions within the S178–R183 region and / or the R188–L194 region of CXCR4. In some such methods, the mutation comprises one or more mutations selected from F189A, N192A, D193A, S178_E179insK, S178_E179insY, S178_E179insR, E179R, D181R, and D182R, and optionally the mutation comprises (1) F189A, N192A, and D193A, (2) S178_E179insK, (3) S178_E179insY, (4) S178_E179insR, or (5) E179R, D181R, and D182R.

[0019] In some such methods, step (c) includes multiple doses of a means for which binding specifically to a second isoform of CXCR4 but not specifically to a first isoform of CXCR4. In some such methods, steps (b) and (c) are performed simultaneously. In some such methods, step (b) is performed before step (c), and if necessary, step (c) is performed after step (b), and includes multiple doses of a means for which binding specifically to a second isoform of CXCR4 but not specifically to a first isoform of CXCR4. In some such methods, step (b) is performed after step (c), and if necessary, step (c) is performed before step (b), and includes multiple doses of a means for which binding specifically to a second isoform of CXCR4 but not specifically to a first isoform of CXCR4. In some such methods, step (c) is performed both before and after step (b), and if necessary, step (c) includes, before step (b), multiple doses of means for which a substance specifically binds to the second isoform of CXCR4 but not specifically to the first isoform of CXCR4, and / or after step (b), multiple doses of means for which a substance specifically binds to the second isoform of CXCR4 but not specifically to the first isoform of CXCR4.

[0020] In some such methods, the donor cells and / or host cells are hematopoietic cells, and, if necessary, the donor cells and / or host cells are immune cells. In some such methods, the donor cells and / or host cells are lymphocytes or lymphoid progenitor cells. In some such methods, the donor cells and / or host cells are T cells. In some such methods, the donor cells and / or host cells are alpha-beta T cells. In some such methods, the donor cells and / or host cells are gamma-delta T cells. In some such methods, the donor cells and / or host cells are tumor-infiltrating lymphocytes (TILs). In some such methods, the donor cells and / or host cells are B cells, and, if necessary, the donor cells and / or host cells are immature B cells, and this method depletes the host's mature B cells from the bone marrow. In some such methods, the donor cells and / or host cells are NK cells. In some such methods, the donor cells and / or host cells are hematopoietic stem and progenitor cells. In some such methods, the donor cells and / or host cells are derived from hematopoietic stem cells or hematopoietic stem and progenitor cells. In some such methods, the donor cells are derived from induced pluripotent stem cells. In some such methods, the subject is a mammal or a non-human mammal, and the donor cells are mammalian cells or non-human mammalian cells. In some such methods, the subject is a human, and the donor cells are human cells.

[0021] In some such methods, the donor cells contain or express a therapeutic molecule. In some such methods, the therapeutic molecule does not target CXCR4. In some such methods, the donor cells contain or express an immunoglobulin, a chimeric antigen receptor (CAR), or an exogenous T cell receptor (TCR). In some such methods, the immunoglobulin, CAR, or exogenous TCR does not target CXCR4.

[0022] In some such methods, the donor cells are autologous. In some such methods, the donor cells are allogeneic or syngeneic. In some such methods, the subject has a disease or disorder, and the method is for treating the disease or disorder in the subject. In some such methods, the subject has cancer, and if necessary, the cancer is a solid tumor carcinoma or a hematological malignancy. In some such methods, the subject has a hematopoietic malignancy, and the method is for treating the hematopoietic malignancy in the subject. In some such methods, the subject has a deficient immune cell or hereditary hematopoietic failure, and if necessary, the hereditary hematopoietic failure is sickle cell disease or severe combined immunodeficiency (SCID).

[0023] In some such methods, the method further includes a step of generating donor cells by modifying a population of cells to express a first isoform of CXCR4 prior to step (a). In some such methods, the population of cells is a population of induced pluripotent stem cells, and the method further includes a step of differentiating the induced pluripotent stem cells into donor cells to be administered in step (a) prior to step (a), and optionally differentiating the induced pluripotent stem cells into hematopoietic cells, lymphocytes or lymphoid progenitor cells, T cells, B cells, NK cells, hematopoietic stem cells, or hematopoietic stem and progenitor cells. In some such methods, the population of cells is a population of hematopoietic stem cells or hematopoietic stem and progenitor cells, and the method further includes a step of differentiating the hematopoietic stem cells or hematopoietic stem and progenitor cells into donor cells to be administered in step (a) prior to step (a), and optionally differentiating the hematopoietic stem cells or hematopoietic stem and progenitor cells into differentiated hematopoietic cells, lymphocytes or lymphoid progenitor cells, T cells, B cells, or NK cells. In some such methods, the step of generating donor cells includes, prior to step (a), introducing an expression vector encoding a first isoform of CXCR4 to express a first isoform of CXCR4, or the step of generating donor cells includes, prior to step (a), editing a genomic locus in a population of cells to express a first isoform of CXCR4. In some such methods, the genomic locus is an endogenous CXCR4 genomic locus. In some such methods, the genomic locus is not an endogenous CXCR4 genomic locus.In some such methods, the editing step includes introducing (1) a nuclease agent or one or more nucleic acids encoding a nuclease agent, wherein the nuclease agent targets a nuclease target sequence at a genomic locus, and (2) an exogenous donor nucleic acid into a population of cells, wherein the nuclease agent cleaves the genomic locus, and the exogenous donor nucleic acid is inserted into or recombined with the genomic locus to produce donor cells expressing a first isoform of CXCR4. In some such methods, the nuclease agent comprises (a) a zinc finger nuclease (ZFN), (b) a transcription activator-like effector nuclease (TALEN), or (c) a guide RNA comprising (i) a Cas protein and (ii) a DNA targeting segment that targets a guide RNA target sequence which is a nuclease target sequence, wherein the guide RNA binds to the Cas protein and directs the Cas protein to the guide RNA target sequence. In some such methods, the nuclease agent comprises a Cas protein and a guide RNA, and optionally the DNA targeting segment comprises a sequence described in any one of SEQ ID NOs: 132-146, or optionally the guide RNA target sequence comprises a sequence described in any one of SEQ ID NOs: 117-131. In some such methods, the Cas protein is a Cas9 protein. In some such methods, the exogenous donor nucleic acid comprises a homology arm. In some such methods, the exogenous donor nucleic acid is a single-stranded oligodeoxynucleotide (ssODN).

[0024] In some such methods, the method further includes the step of isolating a population of cells from or from a different subject prior to the step of modifying the population of cells.

[0025] In another embodiment, combination pharmaceuticals are provided for administration to subjects requiring them. In some such combination pharmaceuticals, the combination pharmaceutical comprises (a) a population of donor cells modified to express a first isoform of CXCR4 different from a second isoform of CXCR4, and (b) a CXCR4 antagonist that specifically binds to the second isoform of CXCR4 but not specifically binds to the first isoform of CXCR4.

[0026] In some such combination therapies, the CXCR4 antagonist selectively inhibits host cells in a target based on the expression of a second isoform of CXCR4. In some such combination therapies, selective inhibition of host cells does not involve ablation of host cells by an active killing mechanism. In some such combination therapies, selective inhibition of host cells includes selective depletion of host cells from the bone marrow. In some such combination therapies, the first and second isoforms are functionally indistinguishable but immunologically distinguishable. In some such combination therapies, donor cells express both the first and second isoforms of CXCR4. In some such combination therapies, donor cells express only the first isoform of CXCR4. In some such combination therapies, the first isoform of CXCR4 is expressed from an expression vector in a population of donor cells, or a genomic locus is edited to express the first isoform of CXCR4 in a population of donor cells. In some such combination drugs, the genomic locus is the endogenous CXCR4 genomic locus. In some such combination drugs, the genomic locus is not the endogenous CXCR4 genomic locus.

[0027] In some such combination drugs, the first isoform of CXCR4 is a genetically engineered isoform of CXCR4. In some such combination drugs, the first isoform of CXCR4 is genetically engineered to contain a mutation that provides an altered epitope, and where necessary, the mutation is an artificial mutation. In some such combination drugs, the altered epitope is located in the binding region of the CXCR4 antagonist, and as a result, the CXCR4 antagonist exhibits reduced or lost ability to bind to and / or inhibit the first isoform of CXCR4 compared to its ability to bind to and / or inhibit the second isoform of CXCR4. In some such combination drugs, both the first and second isoforms of CXCR4 retain the ability to bind to the endogenous ligand, and where necessary, the CXCR4 antagonist blocks the binding of the endogenous ligand to the second isoform of CXCR4 but not to the first isoform of CXCR4. In some such combination drugs, the mutation is located in the extracellular loop 2 (ECL2) region of CXCR4. In some such combination drugs, the mutations include insertions, deletions, or substitutions within the region of CXCR4 from position S178 to R183 and / or within the region of R188 to L194. In some such combination drugs, the mutations include one or more mutations selected from F189A, N192A, D193A, S178_E179insK, S178_E179insY, S178_E179insR, E179R, D181R, and D182R, and as appropriate, the mutations include (1) F189A, N192A, and D193A, (2) S178_E179insK, (3) S178_E179insY, (4) S178_E179insR, or (5) E179R, D181R, and D182R.

[0028] In some such combination pharmaceuticals, the CXCR4 antagonist is an antigen-binding protein. In some such combination pharmaceuticals, the antigen-binding protein is an antibody or its antigen-binding fragment. In some such combination pharmaceuticals, the antigen-binding protein comprises an immunoglobulin light chain or its variable region containing three light chain CDRs and an immunoglobulin heavy chain or its variable region containing three heavy chain CDRs, wherein each of the three light chain CDRs contains, essentially, or comprises a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 15, 17, and 19, and each of the three heavy chain CDRs contains, essentially, or comprises a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 7, 9, and 11. In some such combination pharmaceuticals, the antigen-binding protein comprises an immunoglobulin light chain variable region containing, essentially, or consisting of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 13, and the antigen-binding protein comprises an immunoglobulin heavy chain variable region containing, essentially, or consisting of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 5. In some such combination pharmaceuticals, the immunoglobulin light chain variable region contains, essentially, or consists of the sequence described in SEQ ID NO: 13, and the immunoglobulin heavy chain variable region contains, essentially, or consists of the sequence described in SEQ ID NO: 5.In some such combination pharmaceuticals, the antigen-binding protein comprises an immunoglobulin light chain or its variable region containing three light chain CDRs and an immunoglobulin heavy chain or its variable region containing three heavy chain CDRs, wherein each of the three light chain CDRs contains, essentially, or comprises a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 31, 33, and 35, and each of the three heavy chain CDRs contains, essentially, or comprises a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 23, 25, and 27. In some such combination pharmaceuticals, the antigen-binding protein comprises an immunoglobulin light chain variable region containing, essentially, or consisting of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 29, and the antigen-binding protein comprises an immunoglobulin heavy chain variable region containing, essentially, or consisting of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 21. In some such combination pharmaceuticals, the immunoglobulin light chain variable region contains, essentially, or consists of the sequence described in SEQ ID NO: 29, and the immunoglobulin heavy chain variable region contains, essentially, or consists of the sequence described in SEQ ID NO: 21.

[0029] In some such combination therapies, the donor cells are hematopoietic cells, and, if necessary, immune cells. In some such combination therapies, the donor cells are lymphocytes or lymphoid progenitor cells. In some such combination therapies, the donor cells are T cells. In some such combination therapies, the donor cells are alpha-beta T cells. In some such combination therapies, the donor cells are gamma-delta T cells. In some such combination therapies, the donor cells are tumor-infiltrating lymphocytes (TILs). In some such combination therapies, the donor cells are B cells. In some such combination therapies, the donor cells are NK cells. In some such combination therapies, the donor cells are hematopoietic stem cells or hematopoietic stem and progenitor cells. In some such combination therapies, the donor cells are derived from induced pluripotent stem cells or hematopoietic stem cells or hematopoietic stem and progenitor cells. In some such combination therapies, the subject is a mammal or a non-human mammal, and the donor cells are mammalian cells or non-human mammalian cells. In some such combination drugs, the subject is human, and the donor cells are human cells. In some such combination drugs, the donor cells contain or express a therapeutic molecule. In some such combination drugs, the therapeutic molecule does not target CXCR4. In some such combination drugs, the donor cells contain or express an immunoglobulin, a chimeric antigen receptor (CAR), or an exogenous T cell receptor (TCR). In some such combination drugs, the immunoglobulin, CAR, or exogenous TCR does not target CXCR4. In some such combination drugs, the donor cells are autologous. In some such combination drugs, the donor cells are allogeneic or syngeneic.

[0030] In some such combination drugs, the subject has a disease or disorder, and the combination drug is intended to treat the disease or disorder in the subject. In some such combination drugs, the subject has cancer. In some such combination drugs, the cancer is a solid tumor carcinoma. In some such combination drugs, the cancer is a hematological malignancy. In some such combination drugs, the subject has a hematopoietic malignancy, and the combination drug is intended to treat the hematopoietic malignancy in the subject. In some such combination drugs, the subject has a deficient immune cell or hereditary hematopoietic failure. In some such combination drugs, the hereditary hematopoietic failure is sickle cell disease or severe combined immunodeficiency (SCID).

[0031] Some such combination drugs include (a) a population of donor cells modified to express a first isoform of CXCR4, wherein the first isoform of CXCR4 is different from a second isoform of CXCR4, and (b) means for specifically binding to the second isoform of CXCR4 but not specifically binding to the first isoform of CXCR4.

[0032] In some such combination drugs, the means for specifically binding to a second isoform of CXCR4 but not specifically binding to a first isoform of CXCR4 selectively inhibits host cells in the target based on the expression of the second isoform of CXCR4. In some such combination drugs, selective inhibition of host cells does not involve ablation of host cells by an active killing mechanism. In some such combination drugs, selective inhibition of host cells includes selective depletion of host cells from the bone marrow.

[0033] In some such combination drugs, the first and second isoforms are functionally indistinguishable but immunologically distinguishable. In some such combination drugs, donor cells express both the first and second isoforms of CXCR4. In some such combination drugs, donor cells express only the first isoform of CXCR4. In some such combination drugs, the first isoform of CXCR4 is expressed from an expression vector in a population of donor cells, or a genomic locus is edited to express the first isoform of CXCR4 in a population of donor cells. In some such combination drugs, the genomic locus is the endogenous CXCR4 genomic locus. In some such combination drugs, the genomic locus is not the endogenous CXCR4 genomic locus.

[0034] In some such combination drugs, the first isoform of CXCR4 is a genetically engineered isoform of CXCR4. In some such combination drugs, the first isoform of CXCR4 is genetically engineered to contain a mutation that provides an altered epitope, and where necessary, the mutation is an artificial mutation. In some such combination drugs, the altered epitope is located in the binding region of a means to specifically bind to the second isoform of CXCR4 but not to the first isoform of CXCR4, and as a result, the means to specifically bind to the second isoform of CXCR4 but not to the first isoform of CXCR4 exhibits a reduced or lost ability to bind to and / or inhibit the first isoform of CXCR4 compared to its ability to bind to and / or inhibit the second isoform of CXCR4. In some such combination drugs, both the first and second isoforms of CXCR4 retain the ability to bind to the endogenous ligand, and the means to specifically bind to the second isoform of CXCR4, if necessary, but not specifically to the first isoform of CXCR4, is to block the binding of the endogenous ligand to the second isoform of CXCR4, but not to the first isoform of CXCR4. In some such combination drugs, the mutation is located in the extracellular loop 2 (ECL2) region of CXCR4. In some such combination drugs, the mutation includes insertions, deletions, or substitutions within the S178–R183 region and / or the R188–L194 region of CXCR4. In some such combination drugs, the mutations include one or more mutations selected from F189A, N192A, D193A, S178_E179insK, S178_E179insY, S178_E179insR, E179R, D181R, and D182R, and optionally the mutations include (1) F189A, N192A, and D193A, (2) S178_E179insK, (3) S178_E179insY, (4) S178_E179insR, or (5) E179R, D181R, and D182R.

[0035] In some such combination therapies, the donor cells are hematopoietic cells, and, if necessary, immune cells. In some such combination therapies, the donor cells are lymphocytes or lymphoid progenitor cells. In some such combination therapies, the donor cells are T cells. In some such combination therapies, the donor cells are alpha-beta T cells. In some such combination therapies, the donor cells are gamma-delta T cells. In some such combination therapies, the donor cells are tumor-infiltrating lymphocytes (TILs). In some such combination therapies, the donor cells are B cells. In some such combination therapies, the donor cells are NK cells. In some such combination therapies, the donor cells are hematopoietic stem cells or hematopoietic stem and progenitor cells. In some such combination therapies, the donor cells are derived from induced pluripotent stem cells or hematopoietic stem cells or hematopoietic stem and progenitor cells. In some such combination therapies, the subject is a mammal or a non-human mammal, and the donor cells are mammalian cells or non-human mammalian cells. In some such combination drugs, the target is human, and the donor cells are human cells.

[0036] In some such combination therapies, donor cells contain or express a therapeutic molecule. In some such combination therapies, the therapeutic molecule does not target CXCR4. In some such combination therapies, donor cells contain or express an immunoglobulin, a chimeric antigen receptor (CAR), or an exogenous T cell receptor (TCR). In some such combination therapies, the immunoglobulin, CAR, or exogenous TCR does not target CXCR4.

[0037] In some such combination therapies, the donor cells are autologous. In some such combination therapies, the donor cells are allogeneic or syngeneic. In some such combination therapies, the subject has a disease or disorder, and the combination therapy is intended to treat the disease or disorder in the subject. In some such combination therapies, the subject has cancer, and if necessary, the cancer is a solid tumor or a hematological malignancy. In some such combination therapies, the subject has a hematopoietic malignancy, and the combination therapy is intended to treat the hematopoietic malignancy in the subject. In some such combination therapies, the subject has a deficient immune cell or hereditary hematopoietic failure, and if necessary, the hereditary hematopoietic failure is sickle cell disease or severe combined immunodeficiency (SCID).

[0038] In another embodiment, isolated cells or populations of cells are also provided that have been modified to express a first isoform of CXCR4, distinct from a second isoform of CXCR4. In some such isolated cells or populations of cells, the first isoform of CXCR4 has been genetically engineered to include a mutation that provides an altered epitope, the altered epitope being located in the binding region of a CXCR4 antagonist, so that the CXCR4 antagonist exhibits reduced or lost ability to bind to and / or inhibit the first isoform of CXCR4 compared to its ability to bind to and / or inhibit the second isoform of CXCR4, while the first isoform of CXCR4 retains its binding to its endogenous ligand.

[0039] In some such isolated cells or populations of cells, the mutation is artificial. In some such isolated cells or populations of cells, both the first and second isoforms of CXCR4 retain the ability to bind to the endogenous ligand, and, if necessary, the CXCR4 antagonist blocks the binding of the endogenous ligand to the second isoform of CXCR4 but not to the first isoform of CXCR4. In some such isolated cells or populations of cells, the first and second isoforms are functionally indistinguishable but immunologically distinguishable. In some such isolated cells or populations of cells, one or more cells express both the first and second isoforms of CXCR4. In some such isolated cells or populations of cells, one or more cells express only the first isoform of CXCR4. In some such isolated cells or populations of cells, the first isoform of CXCR4 is expressed in cells(s) from an expression vector, or the genomic locus is edited to express the first isoform of CXCR4 in cells(s). In some such isolated cells or populations of cells, the genomic locus is the endogenous CXCR4 genomic locus. In some such isolated cells or populations of cells, the genomic locus is not the endogenous CXCR4 genomic locus.

[0040] In some such isolated cells or populations of cells, the mutation is located in the extracellular loop 2 (ECL2) region of CXCR4. In some such isolated cells or populations of cells, the mutation includes insertions, deletions, or substitutions within the S178–R183 region and / or the R188–L194 region of CXCR4. In some such isolated cells or populations of cells, the mutations include one or more mutations selected from F189A, N192A, D193A, S178_E179insK, S178_E179insY, S178_E179insR, E179R, D181R, and D182R, and optionally the mutations include (1) F189A, N192A, and D193A, (2) S178_E179insK, (3) S178_E179insY, (4) S178_E179insR, or (5) E179R, D181R, and D182R. In some such isolated cells or populations of cells, the first and second isoforms are immunologically distinguishable by a CXCR4 antagonist, which specifically binds to the second isoform of CXCR4 but not to the first isoform of CXCR4.

[0041] In some such isolated cells or populations of cells, the CXCR4 antagonist is an antigen-binding protein. In some such isolated cells or populations of cells, the antigen-binding protein is an antibody or its antigen-binding fragment. In some such isolated cells or populations of cells, the antigen-binding protein comprises an immunoglobulin light chain or its variable region containing three light chain CDRs and an immunoglobulin heavy chain or its variable region containing three heavy chain CDRs, wherein each of the three light chain CDRs contains, essentially, or consists of, a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 15, 17, and 19, and each of the three heavy chain CDRs contains, essentially, or consists of, a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 7, 9, and 11. In some such isolated cells or populations of cells, the three light chain CDRs contain, are essentially, or consist of, the sequences described in SEQ ID NOs. 15, 17, and 19, respectively, and the three heavy chain CDRs contain, are essentially, or consist of, the sequences described in SEQ ID NOs. 7, 9, and 11, respectively. In some such isolated cells or populations of cells, the antigen-binding protein contains an immunoglobulin light chain variable region containing, is essentially, or consists of a sequence that is at least 90% identical to, is essentially, or consists of, the sequence described in SEQ ID NOs.In some such isolated cells or populations of cells, the antigen-binding protein comprises an immunoglobulin light chain or its variable region containing three light chain CDRs and an immunoglobulin heavy chain or its variable region containing three heavy chain CDRs, wherein each of the three light chain CDRs contains, essentially, or comprises a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 31, 33, and 35, and each of the three heavy chain CDRs contains, essentially, or comprises a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 23, 25, and 27. In some such isolated cells or populations of cells, the antigen-binding protein includes an immunoglobulin light chain variable region containing, essentially, or consisting of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 29, and the antigen-binding protein includes an immunoglobulin heavy chain variable region containing, essentially, or consisting of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 21. In some such isolated cells or populations of cells, the immunoglobulin light chain variable region includes, essentially, or consists of the sequence described in SEQ ID NO: 29, and the immunoglobulin heavy chain variable region includes, essentially, or consists of the sequence described in SEQ ID NO: 21.

[0042] In some such isolated cells or populations of cells, the cells(s) are hematopoietic cells(plural), and, where necessary, immune cells(plural). In some such isolated cells or populations of cells, the cells(s) are lymphocytes or lymphoid progenitor cells(plural). In some such isolated cells or populations of cells, the cells(s) are T cells(plural). In some such isolated cells or populations of cells, the cells(s) are alpha-beta T cells(plural). In some such isolated cells or populations of cells, the cells(s) are gamma-delta T cells(plural). In some such isolated cells or populations of cells, the cells(s) are tumor-infiltrating lymphocytes(plural) (TILs). In some such isolated cells or populations of cells, the cells(s) are B cells(plural). In some such isolated cells or populations of cells, the cell(s) are NK cells. In some such isolated cells or populations of cells, the cell(s) are hematopoietic stem cells or hematopoietic stem and progenitor cells. In some such isolated cells or populations of cells, the cell(s) are induced pluripotent stem cells. In some such isolated cells or populations of cells, the cell(s) are mammalian cells or non-human mammalian cells. In some such isolated cells or populations of cells, the cell(s) are human cells. In some such isolated cells or populations of cells, the cell(s) contain or express a therapeutic molecule. In some such isolated cells or populations of cells, the therapeutic molecule does not target CXCR4. In some such isolated cells or populations of cells, the cell(s) contain or express immunoglobulins, chimeric antigen receptors (CARs), or exogenous T cell receptors (TCRs).In some such isolated cells or populations of cells, immunoglobulins, CARs, or exogenous TCRs do not target CXCR4. In some such isolated cells or populations of cells, the cells(s) are isolated from the subject. In some such isolated cells or populations of cells, the cells(s) are intended for use in the treatment of subjects having cells expressing a second isoform of CXCR4. In some such isolated cells or populations of cells, the cells(s) are isolated from the subject.

[0043] In another embodiment, a method for producing either of the isolated cells or populations of cells described above is provided. Some such methods include the step of modifying the cells or population of cells to express a first isoform of CXCR4.

[0044] In some such methods, the modification step includes introducing an expression vector encoding a first isoform of CXCR4, or the modification step includes editing a genomic locus to express a first isoform of CXCR4. In some such methods, the genomic locus is an endogenous CXCR4 genomic locus. In some such methods, the genomic locus is not an endogenous CXCR4 genomic locus.

[0045] In some such methods, the editing step includes introducing into a cell (1) a nuclease agent or one or more nucleic acids encoding a nuclease agent, wherein the nuclease agent targets a nuclease target sequence at a genomic locus, and (2) an exogenous donor nucleic acid, wherein the nuclease agent cleaves the genomic locus, and the exogenous donor nucleic acid is inserted into or recombined with the genomic locus to produce a cell expressing a first isoform of CXCR4. In some such methods, the nuclease agent includes (a) a zinc finger nuclease (ZFN), (b) a transcription activator-like effector nuclease (TALEN), or (c) a guide RNA comprising (i) a Cas protein and (ii) a guide RNA that binds to the Cas protein and directs the Cas protein to the guide RNA target sequence. In some such methods, the nuclease agent comprises a Cas protein and a guide RNA, and optionally the DNA targeting segment comprises a sequence described in any one of SEQ ID NOs. 132–146, or optionally the guide RNA target sequence comprises a sequence described in any one of SEQ ID NOs. 117–131. In some such methods, the Cas protein is the Cas9 protein. In some such methods, the exogenous donor nucleic acid comprises homology arms. In some such methods, the exogenous donor nucleic acid is a single-stranded oligodeoxynucleotide (ssODN).

[0046] In another embodiment, genetically engineered human CXC chemokine receptor 4 (CXCR4) protein is provided. Some such genetically engineered proteins include artificial mutations that provide an altered epitope, the altered epitope located in the binding region of a CXCR4 antagonist, resulting in the CXCR4 antagonist exhibiting reduced or lost ability to bind to and / or inhibit the genetically engineered human CXCR4 protein compared to its ability to bind to and / or inhibit the wild-type human CXCR4 protein, while the genetically engineered human CXCR4 protein retains its binding to its endogenous ligand(s).

[0047] In some such genetically modified proteins, the genetically modified CXCR4 protein is functionally indistinguishable from the native CXCR4 protein, but immunologically distinguishable.

[0048] In some such genetically engineered proteins, the mutation is located in the extracellular loop 2 (ECL2) region of CXCR4. In some such genetically engineered proteins, the mutation includes insertions, deletions, or substitutions within the S178–R183 region and / or the R188–L194 region of CXCR4. In some such genetically engineered proteins, the mutations include one or more mutations selected from F189A, N192A, D193A, S178_E179insK, S178_E179insY, S178_E179insR, E179R, D181R, and D182R, and, if necessary, the mutations include (1) F189A, N192A, and D193A, (2) S178_E179insK, (3) S178_E179insY, (4) S178_E179insR, or (5) E179R, D181R, and D182R. In some such genetically engineered proteins, the genetically engineered CXCR4 protein and the native CXCR4 protein are functionally indistinguishable by CXCR4 antagonists but immunologically distinguishable.

[0049] In some such genetically engineered proteins, the CXCR4 antagonist is an antigen-binding protein. In some such genetically engineered proteins, the antigen-binding protein is an antibody or its antigen-binding fragment. In some such genetically engineered proteins, the antigen-binding protein comprises an immunoglobulin light chain or its variable region containing three light chain CDRs and an immunoglobulin heavy chain or its variable region containing three heavy chain CDRs, wherein each of the three light chain CDRs contains, is essentially, or comprises a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 15, 17, and 19, and each of the three heavy chain CDRs contains, is essentially, or comprises a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 7, 9, and 11. In some such genetically engineered proteins, the three light chain CDRs contain, are essentially, or consist of, the sequences described in SEQ ID NOs: 15, 17, and 19, respectively, and the three heavy chain CDRs contain, are essentially, or consist of the sequences described in SEQ ID NOs: 7, 9, and 11, respectively. In some such genetically engineered proteins, the antigen-binding protein contains an immunoglobulin light chain variable region containing, is essentially, or consists of a sequence that is at least 90% identical to, is essentially, or consists of, the sequence described in SEQ ID NOs: 13, and the antigen-binding protein contains an immunoglobulin heavy chain variable region containing, is essentially, or consists of a sequence that is at least 90% identical to, is essentially, or consists of, the sequence described in SEQ ID NOs: 5. In some such genetically engineered proteins, the immunoglobulin light chain variable region contains, is essentially, or consists of, the sequence described in SEQ ID NOs: 13, and the immunoglobulin heavy chain variable region contains, is essentially, or consists of, the sequence described in SEQ ID NOs: 5.In some such genetically engineered proteins, the antigen-binding protein comprises an immunoglobulin light chain or its variable region containing three light chain CDRs and an immunoglobulin heavy chain or its variable region containing three heavy chain CDRs, wherein each of the three light chain CDRs contains, essentially, or comprises a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 31, 33, and 35, and each of the three heavy chain CDRs contains, essentially, or comprises a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 23, 25, and 27. In some such genetically engineered proteins, the antigen-binding protein includes an immunoglobulin light chain variable region containing, essentially, or consisting of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 29, and the antigen-binding protein includes an immunoglobulin heavy chain variable region containing, essentially, or consisting of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 21. In some such genetically engineered proteins, the immunoglobulin light chain variable region includes, essentially, or consists of the sequence described in SEQ ID NO: 29, and the immunoglobulin heavy chain variable region includes, essentially, or consists of the sequence described in SEQ ID NO: 21.

[0050] In another embodiment, a nucleic acid is provided which encodes one of the above-mentioned genetically modified human CXCR4 proteins, and optionally is an expression vector encoding a genetically modified human CXCR4 protein.

[0051] In another embodiment, a method is provided for producing a genetically engineered human CXC chemokine receptor type 4 (CXCR4) protein containing an artificial mutation that provides an altered epitope, wherein the altered epitope is located in the binding region of a CXCR4 antagonist, and as a result, the CXCR4 antagonist exhibits reduced or lost ability to bind to and / or inhibit the genetically engineered human CXCR4 protein compared to its ability to bind to and / or inhibit the wild-type human CXCR4 protein, and the genetically engineered human CXCR4 protein retains its binding to its endogenous ligand(s). Some such methods include (a) determining an epitope in the binding region of a CXCR4 antagonist; (b) selecting a site for generating an artificial mutation that provides a modified epitope in the binding region of a CXCR4 antagonist; (c) generating a genetically engineered human CXCR4 protein containing the artificial mutation that provides a modified epitope in the binding region of a CXCR4 antagonist; and (d) testing the genetically engineered human CXCR4 protein to determine whether the CXCR4 antagonist exhibits reduced or lost ability to bind to and / or inhibit the genetically engineered human CXCR4 protein compared to its ability to bind to and / or inhibit the wild-type human CXCR4 protein, and to determine whether the genetically engineered human CXCR4 protein retains binding to its endogenous ligand(s).

[0052] In some such methods, the epitopes of CXCR4 antagonists are determined by alanine scanning mutation analysis, peptide blot analysis, peptide cleavage analysis, crystallographic studies, NMR analysis, epitope excision, epitope extraction, antigen chemical modification, and / or hydrogen / deuterium exchange detected by mass spectrometry. In some such methods, the epitopes of CXCR4 antagonists are determined by high-resolution cryo-electron microscopy analysis of CXCR4 antagonists complexed with human CXCR4 protein. In some such methods, the site of artificial mutation is selected so that it (I) is non-conserved across different mammalian species, (II) does not result in a change in secondary structure, (III) is located in a site accessible to ligand binding, (IV) is not located in a site involved in predicted or experimentally established or confirmed protein-protein interactions, (V) does not result in the deletion or introduction of disulfide bonds, intermolecular or intramolecular interactions, or hydrophobic stacking, (VI) does not result in the deletion or introduction of a post-translational protein modification site, and / or (VII) is located in a site that has a unique topology compared to other mammalian proteins by crystal structure analysis or computer-aided structural prediction.

[0053] In some such methods, the step of generating a genetically engineered human CXCR4 protein includes modifying a cell or population of cells to express the genetically engineered human CXCR4 protein. In some such methods, the modifying step includes introducing an expression vector encoding the genetically engineered human CXCR4 protein, or the modifying step includes editing a genomic locus to express the genetically engineered human CXCR4 protein. In some such methods, the genomic locus is an endogenous CXCR4 genomic locus. In some such methods, the genomic locus is not an endogenous CXCR4 genomic locus. In some such methods, the editing step includes introducing into a cell (1) a nuclease agent or one or more nucleic acids encoding a nuclease agent, wherein the nuclease agent targets a nuclease target sequence at a genomic locus, and (2) an exogenous donor nucleic acid, wherein the nuclease agent cleaves the genomic locus, and the exogenous donor nucleic acid is inserted into or recombined with the genomic locus to produce a cell expressing a genetically engineered human CXCR4 protein. In some such methods, the nuclease agent includes (a) a zinc finger nuclease (ZFN), (b) a transcription activator-like effector nuclease (TALEN), or (c) a guide RNA comprising (i) a Cas protein and (ii) a guide RNA that binds to the Cas protein and directs the Cas protein to the guide RNA target sequence. In some such methods, the nuclease agent comprises a Cas protein and a guide RNA, and optionally the DNA targeting segment comprises a sequence described in any one of SEQ ID NOs. 132–146, or optionally the guide RNA target sequence comprises a sequence described in any one of SEQ ID NOs. 117–131. In some such methods, the Cas protein is the Cas9 protein.In some such methods, the exogenous donor nucleic acid contains homologous arms. In some such methods, the exogenous donor nucleic acid is a single-stranded oligodeoxynucleotide (ssODN).

[0054] In some such methods, genetically engineered human CXCR4 proteins are functionally indistinguishable from native CXCR4 proteins, but immunologically distinguishable.

[0055] In some such methods, the mutation is located in the extracellular loop 2 (ECL2) region of CXCR4. In some such methods, the mutation involves an insertion, deletion, or substitution within the region of CXCR4 from S178 to R183 and / or within the region of R188 to L194. In some such methods, the mutation comprises one or more mutations selected from F189A, N192A, D193A, S178_E179insK, S178_E179insY, S178_E179insR, E179R, D181R, and D182R, and optionally the mutation comprises (1) F189A, N192A, and D193A, (2) S178_E179insK, (3) S178_E179insY, (4) S178_E179insR, or (5) E179R, D181R, and D182R.

[0056] In some such methods, genetically engineered human CXCR4 protein and native CXCR4 protein are functionally indistinguishable by CXCR4 antagonists but immunologically distinguishable. In some such methods, the CXCR4 antagonist is an antigen-binding protein. In some such methods, the antigen-binding protein is an antibody or its antigen-binding fragment. In some such methods, the antigen-binding protein comprises an immunoglobulin light chain or its variable region containing three light chain CDRs and an immunoglobulin heavy chain or its variable region containing three heavy chain CDRs, each containing, essentially, or comprising a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 15, 17, and 19, and each containing, essentially, or comprising a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 7, 9, and 11. In some such methods, the three light chain CDRs each contain, are essentially, or consist of the sequences described in SEQ ID NOs. 15, 17, and 19, and the three heavy chain CDRs each contain, are essentially, or consist of the sequences described in SEQ ID NOs. 7, 9, and 11. In some such methods, the antigen-binding protein includes an immunoglobulin light chain variable region containing, being essentially, or consisting of a sequence that is at least 90% identical to the sequence described in SEQ ID NOs. 13, and the antigen-binding protein includes an immunoglobulin heavy chain variable region containing, being essentially, or consisting of a sequence that is at least 90% identical to the sequence described in SEQ ID NOs. 5. In some such methods, the immunoglobulin light chain variable region contains, is essentially, or consists of the sequence described in SEQ ID NOs. 13, and the immunoglobulin heavy chain variable region contains, is essentially, or consists of the sequence described in SEQ ID NOs.In some such methods, the antigen-binding protein comprises an immunoglobulin light chain or its variable region containing three light chain CDRs and an immunoglobulin heavy chain or its variable region containing three heavy chain CDRs, wherein each of the three light chain CDRs contains, essentially, or comprises a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 31, 33, and 35, and each of the three heavy chain CDRs contains, essentially, or comprises a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 23, 25, and 27. In some such methods, the antigen-binding protein includes an immunoglobulin light chain variable region containing, essentially, or consisting of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 29, and the antigen-binding protein includes an immunoglobulin heavy chain variable region containing, essentially, or consisting of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 21. In some such methods, the immunoglobulin light chain variable region includes, essentially, or consists of the sequence described in SEQ ID NO: 29, and the immunoglobulin heavy chain variable region includes, essentially, or consists of the sequence described in SEQ ID NO: 21.

[0057] Provided herein are methods for the in vivo selective depletion of unedited cells from bone marrow and the repopulation of edited cells in subjects requiring the same; combination pharmaceuticals for administration to subjects requiring the same; isolated cells or populations of cells modified to express a first isoform of CXCR4 different from a second isoform of CXCR4; methods for producing isolated cells or populations of cells, comprising the step of modifying cells or populations of cells to express a first isoform of CXCR4; genetically engineered CXC chemokine receptor 4 (CXCR4) protein, including artificial mutations that provide an altered epitope; and nucleic acids encoding the genetically engineered CXCR4 protein.

[0058] In one embodiment, a method is provided for the in vivo selective depletion of unedited cells from the bone marrow and the regrowth of edited cells in a subject requiring such depletion. Some such methods include the steps of (a) providing edited cells that have been modified to express a first isoform of CXCR4 different from a second isoform of CXCR4, wherein the second isoform is expressed in unedited cells of a subject; (b) administering the edited cells to a subject; and (c) selectively depleting unedited cells from the bone marrow in the subject based on the expression of the second isoform of CXCR4.

[0059] In some such methods, the first and second isoforms are functionally indistinguishable but immunologically distinguishable. In some such methods, edited cells express both the first and second isoforms of CXCR4. In some such methods, edited cells express only the first isoform of CXCR4. In some such methods, the genomic locus is edited to express the first isoform of CXCR4 in edited cells. In some such methods, the genomic locus is the CXCR4 genomic locus. In some such methods, the genomic locus is not the CXCR4 genomic locus.

[0060] In some such methods, the first isoform of CXCR4 is a genetically engineered isoform of CXCR4. In some such methods, the first isoform of CXCR4 is genetically engineered to contain a mutation that provides an altered epitope, and where necessary, the mutation is an artificial mutation. In some such methods, the altered epitope is located in an immunoglobulin light chain or its variable region containing three light chain CDRs and in an antibody binding region containing an immunoglobulin heavy chain or its variable region containing three heavy chain CDRs, where the three light chain CDRs each contain, are essentially derived from, or consist of the sequences described in SEQ ID NOs. 15, 17, and 19, and the three heavy chain CDRs each contain, are essentially derived from, or consist of the sequences described in SEQ ID NOs. 7, 9, and 11. In some such methods, the altered epitope is located in an immunoglobulin light chain or its variable region containing three light chain CDRs and in an antibody binding region containing an immunoglobulin heavy chain or its variable region containing three heavy chain CDRs, wherein the three light chain CDRs each contain, are essentially, or consist of the sequences described in SEQ ID NOs. 31, 33, and 35, respectively, and the three heavy chain CDRs each contain, are essentially, or consist of the sequences described in SEQ ID NOs. 23, 25, and 27. In some such methods, the mutation is located in the extracellular loop 2 (ECL2) region of CXCR4. In some such methods, the mutation includes an insertion, deletion, or substitution within the S178–R183 region and / or the R188–L194 region of CXCR4. In some such methods, the mutation comprises one or more mutations selected from F189A, N192A, D193A, S178_E179insK, S178_E179insY, S178_E179insR, E179R, D181R, and D182R, and optionally the mutation comprises (1) F189A, N192A, and D193A, (2) S178_E179insK, (3) S178_E179insY, (4) S178_E179insR, or (5) E179R, D181R, and D182R.

[0061] In some such methods, selective depletion in step (c) involves administering a CXCR4 antagonist to a target, the CXCR4 antagonist specifically binding to a second isoform of CXCR4 but not to a first isoform of CXCR4. In some such methods, the CXCR4 antagonist is an antigen-binding protein. In some such methods, the antigen-binding protein is an antibody or an antigen-binding fragment thereof. In some such methods, the antigen-binding protein comprises an immunoglobulin light chain or its variable region containing three light chain CDRs and an immunoglobulin heavy chain or its variable region containing three heavy chain CDRs, each containing, essentially, or comprising a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 15, 17, and 19, and each containing, essentially, or comprising a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 7, 9, and 11. In some such methods, the three light chain CDRs each contain, essentially consist of, or comprise the sequences described in SEQ ID NOs. 15, 17, and 19, and the three heavy chain CDRs each contain, essentially consist of, or comprise the sequences described in SEQ ID NOs. 7, 9, and 11. In some such methods, the immunoglobulin light chain or its variable region contains, essentially consists of, or comprises a sequence that is at least 90% identical to the sequence described in SEQ ID NOs. 13, and the immunoglobulin heavy chain or its variable region contains, essentially consists of, or comprises a sequence that is at least 90% identical to the sequence described in SEQ ID NOs.In some such methods, the antigen-binding protein comprises an immunoglobulin light chain or its variable region containing three light chain CDRs and an immunoglobulin heavy chain or its variable region containing three heavy chain CDRs, wherein each of the three light chain CDRs contains, essentially, or comprises a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 31, 33, and 35, and each of the three heavy chain CDRs contains, essentially, or comprises a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 23, 25, and 27. In some such methods, the immunoglobulin light chain or its variable region contains, essentially, or consists of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 29, and the immunoglobulin heavy chain or its variable region contains, essentially, or consists of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 21.

[0062] In some such methods, the edited cells are hematopoietic cells. In some such methods, the edited cells are lymphocytes or lymphoid progenitor cells. In some such methods, the edited cells are T cells. In some such methods, the edited cells are alpha-beta T cells. In some such methods, the edited cells are gamma-delta T cells. In some such methods, the edited cells are tumor-infiltrating lymphocytes (TILs). In some such methods, the edited cells are B cells, and if necessary, the edited cells are immature B cells, and this method depletes unedited mature B cells from the bone marrow. In some such methods, the edited cells are NK cells. In some such methods, the edited cells are hematopoietic stem and progenitor cells. In some such methods, the edited cells are derived from induced pluripotent stem cells. In some such methods, the edited cells are derived from hematopoietic stem cells or hematopoietic stem and progenitor cells. In some such methods, the subject is a mammal or a non-human mammal, and the edited cells are mammalian cells or non-human mammalian cells. In some such methods, the subject is a human, and the edited cells are human cells. In some such methods, the edited cells contain or express therapeutic molecules. In some such methods, the edited cells contain or express immunoglobulins, chimeric antigen receptors (CARs), or exogenous T cell receptors (TCRs). In some such methods, the edited cells are autologous. In some such methods, the edited cells are homogeneous or syngeneic.

[0063] In some such methods, the subject has a hematopoietic malignancy, and the method is for treating the hematopoietic malignancy in the subject. In some such methods, the subject has cancer. In some such methods, the cancer is a blood cancer. In some such methods, the subject has a deficient immune cell or hereditary hematopoietic failure. In some such methods, the hereditary hematopoietic failure is sickle cell disease or severe combined immunodeficiency (SCID).

[0064] In some such methods, steps (b) and (c) are performed simultaneously. In some such methods, step (b) is performed before step (c). In some such methods, step (b) is performed after step (c).

[0065] Some such methods further include a step of generating edited cells by modifying a population of cells to express a first isoform of CXCR4 prior to step (a). In some such methods, the population of cells is a population of induced pluripotent stem cells, and the method further includes a step of differentiating the edited induced pluripotent stem cells into edited cells administered in step (a) prior to step (a), and optionally differentiating the induced pluripotent stem cells into hematopoietic cells, lymphocytes or lymphoid progenitor cells, T cells, B cells, NK cells, hematopoietic stem cells, or hematopoietic stem and progenitor cells. In some such methods, the population of cells is a population of hematopoietic stem cells or hematopoietic stem and progenitor cells, and the method further comprises a step prior to step (a) of differentiating edited hematopoietic stem cells or hematopoietic stem and progenitor cells into edited cells administered in step (a), and, if necessary, differentiating the hematopoietic stem cells or hematopoietic stem and progenitor cells into differentiated hematopoietic cells, lymphocytes or lymphoid progenitor cells, T cells, B cells, or NK cells.

[0066] In some such methods, the step of generating edited cells comprises editing a genomic locus in a population of cells to express a first isoform of CXCR4, prior to step (a). In some such methods, the genomic locus is the CXCR4 genomic locus. In some such methods, the genomic locus is not the CXCR4 genomic locus. In some such methods, the editing step comprises introducing (1) a nuclease or one or more nucleic acids encoding a nuclease, wherein the nuclease targets a nuclease target sequence at the genomic locus, and (2) an exogenous donor nucleic acid into a population of cells, wherein the nuclease cleaves the genomic locus, and the exogenous donor nucleic acid is inserted into or recombined with the genomic locus to generate edited cells expressing a first isoform of CXCR4. In some such methods, the nuclease agent comprises (a) a zinc finger nuclease (ZFN), (b) a transcription activator-like effector nuclease (TALEN), or (c) a guide RNA comprising (i) a Cas protein and (ii) a DNA targeting segment that targets a guide RNA target sequence which is a nuclease target sequence, wherein the guide RNA binds to the Cas protein and directs the Cas protein to the guide RNA target sequence. In some such methods, the nuclease agent comprises a Cas protein and a guide RNA, and optionally the DNA targeting segment comprises a sequence described in any one of SEQ ID NOs: 132-146, or optionally the guide RNA target sequence comprises a sequence described in any one of SEQ ID NOs: 117-131. In some such methods, the Cas protein is a Cas9 protein. In some such methods, the exogenous donor nucleic acid comprises a homology arm. In some such methods, the exogenous donor nucleic acid is a single-stranded oligodeoxynucleotide (ssODN). Some such methods further include the step of isolating a population of cells from one or a different subject, prior to the step of modifying the population of cells.

[0067] In another embodiment, combination pharmaceuticals are provided for administration to subjects requiring them. Some such combination pharmaceuticals include (a) a population of cells modified to express a first isoform of CXCR4 distinct from a second isoform of CXCR4, and (b) a CXCR4 antagonist that specifically binds to the second isoform of CXCR4 but not specifically to the first isoform of CXCR4.

[0068] In some such combination drugs, the first and second isoforms are functionally indistinguishable but immunologically distinguishable. In some such combination drugs, cells express both the first and second isoforms of CXCR4. In some such combination drugs, cells express only the first isoform of CXCR4. In some such combination drugs, the genomic locus is edited to express the first isoform of CXCR4 in a population of cells. In some such combination drugs, the genomic locus is the CXCR4 genomic locus. In some such combination drugs, the genomic locus is not the CXCR4 genomic locus.

[0069] In some such combination drugs, the first isoform of CXCR4 is a genetically engineered isoform of CXCR4. In some such combination drugs, the first isoform of CXCR4 is genetically engineered to contain a mutation that provides an altered epitope, and where necessary, the mutation is an artificial mutation. In some such combination drugs, the altered epitope is located in an immunoglobulin light chain or its variable region containing three light chain CDRs and in an antibody binding region containing an immunoglobulin heavy chain or its variable region containing three heavy chain CDRs, where the three light chain CDRs each contain, are essentially derived from, or consist of the sequences described in SEQ ID NOs. 15, 17, and 19, and the three heavy chain CDRs each contain, are essentially derived from, or consist of the sequences described in SEQ ID NOs. 7, 9, and 11. In some such combination drugs, the altered epitope is located in the binding region of an antibody containing an immunoglobulin light chain or its variable region comprising three light chain CDRs and an immunoglobulin heavy chain or its variable region comprising three heavy chain CDRs, wherein the three light chain CDRs each contain, are essentially, or consist of the sequences described in SEQ ID NOs. 31, 33, and 35, respectively, and the three heavy chain CDRs each contain, are essentially, or consist of the sequences described in SEQ ID NOs. 23, 25, and 27. In some such combination drugs, the mutation is located in the extracellular loop 2 (ECL2) region of CXCR4. In some such combination drugs, the mutation includes insertions, deletions, or substitutions within the S178–R183 region and / or the R188–L194 region of CXCR4. In some such combination drugs, the mutations include one or more mutations selected from F189A, N192A, D193A, S178_E179insK, S178_E179insY, S178_E179insR, E179R, D181R, and D182R, and optionally the mutations include (1) F189A, N192A, and D193A, (2) S178_E179insK, (3) S178_E179insY, (4) S178_E179insR, or (5) E179R, D181R, and D182R.

[0070] In some such combination pharmaceuticals, the CXCR4 antagonist is an antigen-binding protein. In some such combination pharmaceuticals, the antigen-binding protein is an antibody or its antigen-binding fragment. In some such combination pharmaceuticals, the antigen-binding protein comprises an immunoglobulin light chain or its variable region containing three light chain CDRs and an immunoglobulin heavy chain or its variable region containing three heavy chain CDRs, wherein each of the three light chain CDRs contains, essentially, or comprises a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 15, 17, and 19, and each of the three heavy chain CDRs contains, essentially, or comprises a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 7, 9, and 11. In some such combination pharmaceuticals, the immunoglobulin light chain or its variable region contains, is essentially, or consists of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 13, and the immunoglobulin heavy chain or its variable region contains, is essentially, or consists of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 5.In some such combination pharmaceuticals, the antigen-binding protein comprises an immunoglobulin light chain or its variable region containing three light chain CDRs and an immunoglobulin heavy chain or its variable region containing three heavy chain CDRs, wherein each of the three light chain CDRs contains, essentially, or comprises a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 31, 33, and 35, and each of the three heavy chain CDRs contains, essentially, or comprises a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 23, 25, and 27. In some such combination pharmaceuticals, the immunoglobulin light chain or its variable region contains, is essentially, or consists of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 29, and the immunoglobulin heavy chain or its variable region contains, is essentially, or consists of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 21.

[0071] In some such combination therapies, the cells are hematopoietic cells. In some such combination therapies, the cells are lymphocytes or lymphoid progenitor cells. In some such combination therapies, the cells are T cells. In some such combination therapies, the cells are alpha-beta T cells. In some such combination therapies, the cells are gamma-delta T cells. In some such combination therapies, the cells are tumor-infiltrating lymphocytes (TILs). In some such combination therapies, the cells are B cells. In some such combination therapies, the cells are NK cells. In some such combination therapies, the cells are hematopoietic stem cells or hematopoietic stem and progenitor cells. In some such combination therapies, the cells are derived from induced pluripotent stem cells or hematopoietic stem cells or hematopoietic stem and progenitor cells. In some such combination therapies, the subject is a mammal or a non-human mammal, and the cells are mammalian cells or non-human mammalian cells. In some such combination therapies, the subject is a human, and the cells are human cells. In some such combination therapies, the cells contain or express therapeutic molecules. In some such combination drugs, the cells contain or express immunoglobulins, chimeric antigen receptors (CARs), or exogenous T cell receptors (TCRs). In some such combination drugs, the cells are autologous. In some such combination drugs, the cells are homogeneous or syngeneic.

[0072] In some such combination therapies, the subject has a hematopoietic malignancy, and the combination therapy is intended to treat the hematopoietic malignancy in the subject. In some such combination therapies, the subject has cancer. In some such combination therapies, the cancer is a blood cancer. In some such combination therapies, the subject has a deficient immune cell or hereditary hematopoietic deficiency. In some such combination therapies, the hereditary hematopoietic deficiency is sickle cell disease or severe combined immunodeficiency (SCID).

[0073] In another embodiment, an isolated cell or population of cells is provided that has been modified to express a first isoform of CXCR4, distinct from a second isoform of CXCR4.

[0074] In some such isolated cells or populations of cells, the first and second isoforms are functionally indistinguishable but immunologically distinguishable. In some such isolated cells or populations of cells, one or more cells express both the first and second isoforms of CXCR4. In some such isolated cells or populations of cells, one or more cells express only the first isoform of CXCR4. In some such isolated cells or populations of cells, the first isoform of CXCR4 is a genetically engineered isoform of CXCR4. In some such isolated cells or populations of cells, the first isoform of CXCR4 is genetically engineered to contain a mutation that provides an altered epitope, and where necessary, the mutation is an artificial mutation. In some such isolated cells or populations of cells, a genomic locus is edited to express the first isoform of CXCR4 in one or more cells. In some such isolated cells or populations of cells, the genomic locus is the CXCR4 genomic locus. In some such isolated cells or populations of cells, the genomic locus is not the CXCR4 genomic locus.

[0075] In some such isolated cells or populations of cells, the cell(s) In some such isolated cells or populations of cells, the nuclease agent comprises a Cas protein and guide RNA, and optionally the DNA targeting segment comprises a sequence described in any one of SEQ ID NOs. 132–146, or optionally the guide RNA target sequence comprises a sequence described in any one of SEQ ID NOs. 117–131. In some such isolated cells or populations of cells, the Cas protein is the Cas9 protein. In some such isolated cells or populations of cells, the exogenous donor nucleic acid comprises a homology arm. In some such isolated cells or populations of cells, the exogenous donor nucleic acid is a single-stranded oligodeoxynucleotide (ssODN).

[0076] In some such isolated cells or populations of cells, the altered epitopes are located in the binding region of an antibody containing an immunoglobulin light chain or its variable region comprising three light chain CDRs and an immunoglobulin heavy chain or its variable region comprising three heavy chain CDRs, wherein the three light chain CDRs each contain, are essentially, or consist of the sequences described in SEQ ID NOs. 15, 17, and 19, respectively, and the three heavy chain CDRs each contain, are essentially, or consist of the sequences described in SEQ ID NOs. 7, 9, and 11. In some such isolated cells or populations of cells, the altered epitopes are located in the immunoglobulin light chain or its variable region containing three light chain CDRs and in the antibody binding region containing the immunoglobulin heavy chain or its variable region containing three heavy chain CDRs, wherein the three light chain CDRs each contain, are essentially, or consist of the sequences described in SEQ ID NOs. 31, 33, and 35, respectively, and the three heavy chain CDRs each contain, are essentially, or consist of the sequences described in SEQ ID NOs. 23, 25, and 27. In some such isolated cells or populations of cells, the mutations are located in the extracellular loop 2 (ECL2) region of CXCR4. In some such isolated cells or populations of cells, the mutations include insertions, deletions, or substitutions within the S178–R183 region and / or the R188–L194 region of CXCR4. In some such isolated cells or populations of cells, the mutations include one or more mutations selected from F189A, N192A, D193A, S178_E179insK, S178_E179insY, S178_E179insR, E179R, D181R, and D182R, and optionally the mutations include (1) F189A, N192A, and D193A, (2) S178_E179insK, (3) S178_E179insY, (4) S178_E179insR, or (5) E179R, D181R, and D182R.

[0077] In some such isolated cells or populations of cells, the first and second isoforms are functionally indistinguishable but immunologically distinguishable by the CXCR4 antagonist. In some such isolated cells or populations of cells, the CXCR4 antagonist is an antigen-binding protein. In some such isolated cells or populations of cells, the antigen-binding protein is an antibody or its antigen-binding fragment. In some such isolated cells or populations of cells, the antigen-binding protein comprises an immunoglobulin light chain or its variable region containing three light chain CDRs and an immunoglobulin heavy chain or its variable region containing three heavy chain CDRs, each containing, essentially, or comprising a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 15, 17, and 19, and each containing, essentially, or comprising a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 7, 9, and 11. In some such isolated cells or populations of cells, the three light chain CDRs contain, are essentially, or consist of the sequences described in SEQ ID NOs. 15, 17, and 19, respectively, and the three heavy chain CDRs contain, are essentially, or consist of the sequences described in SEQ ID NOs. 7, 9, and 11, respectively. In some such isolated cells or populations of cells, the immunoglobulin light chain or its variable region contains, is essentially, or consists of a sequence that is at least 90% identical to the sequence described in SEQ ID NOs. 13, and the immunoglobulin heavy chain or its variable region contains, is essentially, or consists of a sequence that is at least 90% identical to the sequence described in SEQ ID NOs.In some such isolated cells or populations of cells, the antigen-binding protein comprises an immunoglobulin light chain or its variable region containing three light chain CDRs and an immunoglobulin heavy chain or its variable region containing three heavy chain CDRs, wherein each of the three light chain CDRs contains, essentially, or comprises a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 31, 33, and 35, and each of the three heavy chain CDRs contains, essentially, or comprises a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 23, 25, and 27. In some such isolated cells or populations of cells, the immunoglobulin light chain or its variable region contains, is essentially, or consists of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 29, and the immunoglobulin heavy chain or its variable region contains, is essentially, or consists of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 21.

[0078] In some such isolated cells or populations of cells, the cell(s) are hematopoietic cells(plural). In some such isolated cells or populations of cells, the cell(s) are lymphocytes or lymphoid progenitor cells(plural). In some such isolated cells or populations of cells, the cell(s) are T cells(plural). In some such isolated cells or populations of cells, the cell(s) are alpha-beta T cells(plural). In some such isolated cells or populations of cells, the cell(s) are gamma-delta T cells(plural). In some such isolated cells or populations of cells, the cell(s) are tumor-infiltrating lymphocytes(plural) (TILs). In some such isolated cells or populations of cells, the cell(s) are B cells(plural). In some such isolated cells or populations of cells, the cell(s) are NK cells(plural). In some such isolated cells or populations of cells, the cells(s) are hematopoietic stem cells(or hematopoietic stem and progenitor cells). In some such isolated cells or populations of cells, the cells(s) are induced pluripotent stem cells(or induced pluripotent stem cells). In some such isolated cells or populations of cells, the cells(s) are mammalian cells(or non-human mammalian cells). In some such isolated cells or populations of cells, the cells(s) are human cells(or human cells). In some such isolated cells or populations of cells, the cells(s) contain or express therapeutic molecules. In some such isolated cells or populations of cells, the cells(s) contain or express immunoglobulins, chimeric antigen receptors (CARs), or exogenous T cell receptors (TCRs). In some such isolated cells or populations of cells, the cells(s) are isolated from the subject.

[0079] Some of these isolated cells or populations of cells are intended for use in the treatment of subjects having cells expressing a second isoform of CXCR4. In some of these isolated cells or populations of cells, the cells (one or more) are isolated from the subject.

[0080] In another embodiment, a method is provided for producing either of the isolated cells or populations of cells described above, comprising the step of modifying the cells or population of cells to express a first isoform of CXCR4. In some such methods, the modification step includes editing a genomic locus to express a first isoform of CXCR4. In some such methods, the genomic locus is the CXCR4 genomic locus. In some such methods, the genomic locus is not the CXCR4 genomic locus.

[0081] In some such methods, the editing step involves introducing into a cell (1) a nuclease agent or one or more nucleic acids encoding a nuclease agent, wherein the nuclease agent targets a nuclease target sequence at a genomic locus, and (2) an exogenous donor nucleic acid, wherein the nuclease agent cleaves the genomic locus, and the exogenous donor nucleic acid is inserted into or recombined with the genomic locus to produce an edited cell expressing a first isoform of CXCR4. In some such methods, the nuclease agent comprises (a) a zinc finger nuclease (ZFN), (b) a transcription activator-like effector nuclease (TALEN), or (c) a guide RNA comprising (i) a Cas protein and (ii) a DNA targeting segment that targets a guide RNA target sequence which is a nuclease target sequence, wherein the guide RNA binds to the Cas protein and directs the Cas protein to the guide RNA target sequence. In some such methods, the nuclease agent comprises a Cas protein and a guide RNA, and optionally the DNA targeting segment comprises a sequence described in any one of SEQ ID NOs: 132-146, or optionally the guide RNA target sequence comprises a sequence described in any one of SEQ ID NOs: 117-131. In some such methods, the Cas protein is a Cas9 protein. In some such methods, the exogenous donor nucleic acid comprises a homology arm. In some such methods, the exogenous donor nucleic acid is a single-stranded oligodeoxynucleotide (ssODN).

[0082] In another embodiment, a genetically engineered CXC chemokine receptor type 4 (CXCR4) protein is provided, which includes an artificial mutation that provides an altered epitope. In some such genetically engineered CXCR4 proteins, the genetically engineered CXCR4 protein is functionally indistinguishable from the native CXCR4 protein but immunologically distinguishable. In some such genetically engineered CXCR4 proteins, the altered epitope is located in an immunoglobulin light chain or its variable region containing three light chain CDRs and in an antibody binding region containing an immunoglobulin heavy chain or its variable region containing three heavy chain CDRs, wherein the three light chain CDRs each contain, are essentially derived from, or consist of the sequences described in SEQ ID NOs. 15, 17, and 19, respectively, and the three heavy chain CDRs each contain, are essentially derived from, or consist of the sequences described in SEQ ID NOs. 7, 9, and 11, respectively. In some such genetically engineered CXCR4 proteins, the altered epitopes are located in the immunoglobulin light chain or its variable region containing three light chain CDRs and in the antibody-binding region containing the immunoglobulin heavy chain or its variable region containing three heavy chain CDRs, where the three light chain CDRs each contain, are essentially, or consist of the sequences described in SEQ ID NOs. 31, 33, and 35, respectively, and the three heavy chain CDRs each contain, are essentially, or consist of the sequences described in SEQ ID NOs. 23, 25, and 27, respectively. In some such genetically engineered CXCR4 proteins, the mutations are located in the extracellular loop 2 (ECL2) region of CXCR4. In some such genetically engineered CXCR4 proteins, the mutations include insertions, deletions, or substitutions within the S178–R183 region and / or the R188–L194 region of CXCR4.In some such genetically engineered CXCR4 proteins, the mutations include one or more mutations selected from F189A, N192A, D193A, S178_E179insK, S178_E179insY, S178_E179insR, E179R, D181R, and D182R, and optionally the mutations include (1) F189A, N192A, and D193A, (2) S178_E179insK, (3) S178_E179insY, (4) S178_E179insR, or (5) E179R, D181R, and D182R.

[0083] In some such genetically engineered CXCR4 proteins, the genetically engineered CXCR4 protein and the native CXCR4 protein are functionally indistinguishable by the CXCR4 antagonist but immunologically distinguishable. In some such genetically engineered CXCR4 proteins, the CXCR4 antagonist is an antigen-binding protein. In some such genetically engineered CXCR4 proteins, the antigen-binding protein is an antibody or its antigen-binding fragment. In some such genetically engineered CXCR4 proteins, the antigen-binding protein comprises an immunoglobulin light chain or its variable region containing three light chain CDRs and an immunoglobulin heavy chain or its variable region containing three heavy chain CDRs, each containing, essentially, or comprising a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 15, 17, and 19, and each containing, essentially, or comprising a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 7, 9, and 11. In some such genetically engineered CXCR4 proteins, the three light chain CDRs contain, are essentially, or consist of the sequences described in SEQ ID NOs. 15, 17, and 19, respectively, and the three heavy chain CDRs contain, are essentially, or consist of the sequences described in SEQ ID NOs. 7, 9, and 11, respectively. In some such genetically engineered CXCR4 proteins, the immunoglobulin light chain or its variable region contains, is essentially, or consists of a sequence that is at least 90% identical to the sequence described in SEQ ID NO. 13, and the immunoglobulin heavy chain or its variable region contains, is essentially, or consists of a sequence that is at least 90% identical to the sequence described in SEQ ID NO. 5.In some such genetically engineered CXCR4 proteins, the immunoglobulin light chain or its variable region contains, is essentially, or consists of the sequence described in SEQ ID NO: 13, and the immunoglobulin heavy chain or its variable region contains, is essentially, or consists of the sequence described in SEQ ID NO: 5. In some such genetically engineered CXCR4 proteins, the antigen-binding protein contains an immunoglobulin light chain or its variable region comprising three light chain CDRs and an immunoglobulin heavy chain or its variable region comprising three heavy chain CDRs, each containing, is essentially, or consists of a sequence that is at least 90% identical to the sequences described in SEQ ID NOs: 31, 33, and 35, and each containing, is essentially, or consists of a sequence that is at least 90% identical to the sequences described in SEQ ID NOs: 23, 25, and 27. In some such genetically engineered CXCR4 proteins, the three light chain CDRs each contain, are essentially derived from, or consist of the sequences described in SEQ ID NOs. 31, 33, and 35, respectively, and the three heavy chain CDRs each contain, are essentially derived from, or consist of the sequences described in SEQ ID NOs. 23, 25, and 27, respectively. In some such genetically engineered CXCR4 proteins, the immunoglobulin light chain or its variable region contains, is essentially derived from, or consists of a sequence that is at least 90% identical to the sequence described in SEQ ID NOs. 29, and the immunoglobulin heavy chain or its variable region contains, is essentially derived from, or consists of a sequence that is at least 90% identical to the sequence described in SEQ ID NOs. 21. In some such genetically engineered CXCR4 proteins, the immunoglobulin light chain or its variable region contains, is essentially derived from, or consists of the sequence described in SEQ ID NOs. 29, and the immunoglobulin heavy chain or its variable region contains, is essentially derived from, or consists of the sequence described in SEQ ID NOs. 21.

[0084] In some such genetically modified CXCR4 proteins, the CXCR4 protein is the human CXCR4 protein.

[0085] In another embodiment, nucleic acids encoding any of the above-described genetically modified CXCR4 proteins are provided. [Brief explanation of the drawing]

[0086] [Figure 1A] A schematic diagram of the CXC motif chemokine receptor 4 (CXCR4) antibody-resistant modified receptor (ARMoR) strategy. [Figure 1B] Same as above.

[0087] [Figure 2A] Administration of anti-human CXCR4 (REGN7663, REGN7664) induces leukocyte recruitment in CXCR4-humanized mice. Figure 2A shows a schematic diagram of the experiment, and Figure 2B shows the experimental data. [Figure 2B] Same as above.

[0088] [Figure 3A] Recruitment of hematopoietic progenitor cells in CXCR4 humanized mice after five consecutive administrations of anti-CXCR4 REGN7664. Figure 3A shows a schematic diagram of the experiment, and Figure 3B shows the experimental data. [Figure 3B] Same as above.

[0089] [Figure 4A] In vivo blockade of CXCR4 with anti-CXCR4 agents strongly reduces recirculating mature B cells in the bone marrow (BM). Figure 4A shows a schematic diagram of the experiment, and Figure 4B shows the experimental data. [Figure 4B] Same as above.

[0090] [Figure 5] The effects of in vivo CXCR4 blockade on BM resident maturation and precursor B cells.

[0091] [Figure 6] Human / mouse CXCR4 domain exchange chimeric constructs designed to test the binding site of anti-human CXCR4 antibodies.

[0092] [Figure 7A] The anti-human CXCR4 monoclonal antibodies (mAbs) REGN7663 and REGN7664 bind to the extracellular loop 2 (ECL2) domain of CXCR4. [Figure 7B] Same as above. [Figure 7C] Same as above. [Figure 7D] Same as above.

[0093] [Figure 8A] Stable cell lines expressing domain-exchange chimeric constructs confirm the binding of REGN7663 and REGN7664 to the ECL2 region of human CXCR4. [Figure 8B] Same as above. [Figure 8C-D] Same as above.

[0094] [Figure 9] Modifications within the human CXCR4 ECL2 region designed to map the binding determinants against CXCR4 REGN7663 and REGN7664.

[0095] [Figure 10A] Determinants for the binding of anti-human CXCR4 REGN7663 and REGN7664 in the ECL2 domain. [Figure 10B] Same as above. [Figure 10C] Same as above. [Figure 10D] Same as above.

[0096] [Figure 11] Schematic diagram of a bioassay on CXCR4 signaling function in response to ligand CXC motif chemokine 12 (CXCL12).

[0097] [Figure 12A] Anti-CXCR4 binding resistant CXCR4 variants retain signaling function in response to ligand CXCL12. [Figure 12B] Same as above. [Figure 12C] Same as above.

[0098] [Figure 13-1] Schematic diagram of a bioassay on the blockade of CXCR4 signaling function by anti-CXCR4 mAbs. [Figure 13-2] Same as above.

[0099] [Figure 14A] CXCR4 variants lacking REGN7664 binding are resistant to REGN7664-mediated signaling blockade. [Figure 14B] Same as above.

[0100] [Figure 15] An additional CXCR4 variant with modifications in the established binding region.

[0101] [Figure 16A] Binding of anti-CXCR4 to additional human CXCR4 variants. [Figure 16B] Same as above.

[0102] [Figure 17A] Reconfirmation of the binding patterns of anti-CXCR4 REGN7663 and REGN7664 to selected CXCR4 ARMoR variants in 293.CRE.Luc.CXCR4.KO bioassay cells. [Figure 17B] Same as above.

[0103] [Figure 18] Antibody-resistant CXCR4 variants with minimal modifications retain their signaling function in response to CXCL12. [Modes for carrying out the invention]

[0104] definition The terms “protein,” “polypeptide,” and “peptide” are used interchangeably herein and include polymeric forms of amino acids of any length, including encoded and unencoded amino acids, as well as chemically or biochemically modified or derivatized amino acids. The term also includes modified polymers, such as polypeptides having a modified peptide backbone. The term “domain” refers to any portion of a protein or polypeptide having a particular function or structure.

[0105] Proteins are said to have an "N-terminus" (amino terminus) and a "C-terminus" (carboxyl terminus). The term "N-terminus" refers to the starting end of a protein or polypeptide and ends with an amino acid that has a free amine group (-NH2). The term "C-terminus" refers to the ending end of an amino acid chain (protein or polypeptide) and ends with a free carboxyl group (-COOH).

[0106] The terms “nucleic acid” and “polynucleotide” are used interchangeably herein and include polymeric forms of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, or analogs or modified versions thereof. These include single-stranded, double-stranded, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers containing purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, unnatural, or derivatized nucleotide bases.

[0107] Nucleic acids are said to have a "5' end" and a "3' end" because mononucleotides react in such a way that the 5' phosphate of one mononucleotide pentose ring is unidirectionally bonded to the nearby 3' oxygen via a phosphodiester bond to produce oligonucleotides. The end of an oligonucleotide is called the "5' end" if its 5' phosphate is not bonded to the 3' oxygen of another mononucleotide pentose ring. The end of an oligonucleotide is called the "3' end" if its 3' oxygen is not bonded to the 5' phosphate of another mononucleotide pentose ring. A nucleic acid sequence can be said to have a 5' end and a 3' end even if it is inside a larger oligonucleotide. In both linear and cyclic DNA molecules, individual elements are referred to as the "downstream" or "upstream" or 5' element of the 3' element.

[0108] The terms “expression vector,” “expression construct,” or “expression cassette” refer to recombinant nucleic acids containing a desired coding sequence operably ligated to appropriate nucleic acid sequences required for the expression of a coding sequence operably ligated in a particular host cell or organism. Nucleic acid sequences required for expression in prokaryotes typically include promoters, operators (if necessary), and ribosome binding sites, as well as other sequences. Eukaryotic cells are generally known to utilize promoters, enhancers, and termination and polyadenylation signals, although some elements may be omitted and others added without sacrificing desired expression.

[0109] A “promoter” is a regulatory region of DNA that typically contains a TATA box capable of directing RNA polymerase II to initiate RNA synthesis at the appropriate transcription start site for a particular polynucleotide sequence. Generally, a “promoter” or “promoter sequence” is a regulatory region of DNA that can bind to RNA polymerase in a cell (e.g., directly or through other proteins or substances bound to the promoter) and initiate transcription of a coding sequence. Promoters can be operably ligated to other expression regulatory sequences, including enhancer and repressor sequences, and / or to the polynucleotides of the present invention.Promoters that may be used to control gene expression include the cytomegalovirus (CMV) promoter (U.S. Patent Nos. 5,385,839 and 5,168,062, each of which is incorporated herein by reference in its entirety for all purposes), the SV40 early promoter region (Benoist et al. (1981) Nature 290:304-310, which is incorporated herein by reference in its entirety for all purposes), the promoter contained in the 3' long-term repeat of Roussarcoma virus (Yamamoto et al. (1980) Cell 22:787-797, which is incorporated herein by reference in its entirety for all purposes), the herpesthymidine kinase promoter (Wagner et al. (1981) Proc. Natl. Acad. Sci. USA 78:1441-1445, which is incorporated herein by reference in its entirety for all purposes), and the regulatory sequence of the metallothionein gene (Brinster et al. (1982) Nature). Examples of such materials include, but are not limited to, the Gal4 promoter, the ADC (alcohol dehydrogenase) promoter, the PGK (phosphoglycerol kinase) promoter, or the alkaline phosphatase promoter, e.g., beta-lactamase promoter (Villa-Komaroff et al. (1978) Proc. Natl. Acad. Sci. USA 75:3727-3731, e.g., the whole of which is incorporated herein by reference for all purposes), or tac promoter (DeBoer et al. (1983) Proc. Natl. Acad. Sci. USA 80:21-25, see also “Useful proteins from recombinant bacteria” in Scientific American (1980) 242:74-94, each of which is incorporated herein by reference for all purposes), as well as promoter elements derived from yeast or other fungi, e.g., Gal4 promoter, ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, or alkaline phosphatase promoter.

[0110] In some embodiments, the promoter may additionally include other regions that affect the transcription initiation rate. The promoter sequence modulates the transcription of operably linked polynucleotides. The promoter may be active in one or more cell types (e.g., eukaryotic cells, non-human mammalian cells, human cells, rodent cells, pluripotent cells, one-cell stage embryos, differentiated cells, or combinations thereof). The promoter may be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a temporally restricted promoter (e.g., a developmentally regulatory promoter, but not limited to these), or a spatially restricted promoter (e.g., a cell-specific or tissue-specific promoter, but not limited to these).

[0111] "Operatable linkage" or "operably linked" means the juxtaposition of two or more components (e.g., a promoter and another sequence element) such that both components may function properly and mediate a function exerted by at least one of the other components. In a non-limiting example, a promoter may be operatically linked to a coding sequence if the promoter controls the transcription level of the coding sequence in response to the presence or absence of one or more transcription regulators. An operatable linkage may include such sequences acting adjacent to or in trans (e.g., a regulatory sequence may act at a distance to control the transcription of a coding sequence, but is not limited to this). A polynucleotide encoding a polypeptide is "operably linked" to a promoter or other expression regulatory sequence if, in a cell or other expression system, the sequence directs the transcription of the coding sequence to RNA, preferably mRNA, mediated by RNA polymerase, which can then undergo RNA splicing (if introns are present) and, if necessary, be translated into the protein encoded by the coding sequence.

[0112] In relation to proteins, nucleic acids, and cells, the term “isolated” includes proteins, nucleic acids, and cells that are purified relative to other cellular or biological components that may normally be present in situ, and at most includes substantially pure preparations of proteins, nucleic acids, or cells. “Isolated” antigen-binding proteins (e.g., antibodies or their antigen-binding fragments), polypeptides, polynucleotides, and vectors do not contain, at least partially, other biological molecules derived from the cells or cell cultures in which they are produced. Such biological molecules include nucleic acids, proteins, other antibodies or antigen-binding fragments, lipids, carbohydrates, or other materials, e.g., cell debris and growth media. Isolated antigen-binding proteins may also not contain, at least partially, components of the expression system, e.g., biological molecules derived from the host cells or their growth media. In general, the term “isolated” is not intended to refer to the complete absence of such biological molecules (e.g., small or insignificant amounts of impurities may remain), the absence of water, buffers, or salts, or components of pharmaceutical formulations containing antigen-binding proteins (e.g., antibodies or antigen-binding fragments).

[0113] In some embodiments, the term “isolated” may include proteins and nucleic acids that do not have naturally occurring counterparts, or proteins or nucleic acids that are chemically synthesized and therefore substantially free from other proteins or nucleic acids. The term “isolated” may include proteins, nucleic acids, or cells that have been separated or purified from most other cellular or biological components that naturally accompany them (e.g., other cellular proteins, nucleic acids, or cellular or extracellular components, but not limited to these).

[0114] "Codon optimization" is a process that utilizes codon degeneracy, which is manifested by the multiplicity of three-base-pair codon combinations that specify amino acids, to modify nucleic acid sequences to enhance expression in a particular host cell, generally by replacing at least one codon in the original sequence with a codon that is more or most frequently used in the host cell's gene, while maintaining the original amino acid sequence. As a non-limiting example, a nucleic acid encoding a protein may be modified to replace codons that are more frequently used in a given prokaryotic or eukaryotic cell, including bacterial cells, yeast cells, human cells, non-human cells, mammalian cells, rodent cells, mouse cells, rat cells, hamster cells, or any other host cell, compared to a naturally occurring nucleic acid sequence. Codon usage tables are readily available, for example, in "codon usage databases." These tables may be adapted in several formats. See Nakamura et al. (2000) Nucleic Acids Res. 28(1):292, which is incorporated herein by reference in its entirety for all purposes. Computer algorithms for codon optimization of specific sequences for expression in specific hosts are also available (see, for example, Gene Forge).

[0115] The term “locus” refers to a specific location on a chromosome in the genome of an organism of a gene (or important sequence), DNA sequence, polypeptide coding sequence, or position. As a non-limiting example, “CXCR4 locus” may refer to a specific location on a chromosome in the genome of an organism of a CXCR4 gene, CXCR4 DNA sequence, CXCR4 protein coding sequence, or CXCR4 position, as identified with respect to the location where such sequence exists. “CXCR4 locus” may, as a non-limiting example, include regulatory elements of the CXCR4 gene, such as enhancers, promoters, 5' and / or 3' untranslated regions (UTRs), or combinations thereof.

[0116] The term “gene” refers to a DNA sequence within a chromosome that, in its natural state, may include at least one coding region and at least one non-coding region. The DNA sequence within a chromosome that codes for a product (e.g., RNA products and / or polypeptide products) may include a coding region interrupted by non-coding introns, as well as sequences located adjacent to the coding region at both the 5' and 3' ends, so that the gene corresponds to full-length mRNA (including the 5' and 3' untranslated sequences). In addition, other non-coding sequences, including regulatory sequences (e.g., promoters, enhancers, and transcription factor binding sites), polyadenylation signals, internal ribosome entry sites, silencers, insulator sequences, and matrix-binding regions, may be present in the gene. These sequences may be located near the coding region of the gene (e.g., within 10 kb, but not limited to this) or distally, and they may affect the level or rate of transcription and translation of the gene.

[0117] The term "allele" refers to a variant form of a gene. Some genes have various different forms, and these are located at the same position on a chromosome, or at a specific genetic locus. Diploid organisms have two alleles at each genetic locus. Each allele pair represents the genotype of a particular genetic locus. A genotype is described as homozygous if two identical alleles are present at a particular locus, and as heterozygous if the two alleles are different.

[0118] The term "wild-type" includes entities that have the structure (e.g., nucleotide or amino acid sequence) as they would be found in their normal state or context (as opposed to variants, diseased or altered). Wild-type genes and polypeptides often exist in multiple different forms (e.g., alleles).

[0119] The term "variant" refers to a nucleotide sequence that differs (for example, by a single nucleotide, but not limited to) from the most widespread sequence in a population, or a protein sequence that differs (for example, by a single amino acid, but not limited to) from the most widespread sequence in a population.

[0120] The term "fragment," when referring to a protein, means a protein that is shorter than or has fewer amino acids than the full-length protein. The term "fragment," when referring to a nucleic acid, means a nucleic acid that is shorter than or has fewer nucleotides than the full-length nucleic acid. Non-limiting examples of protein fragments include N-terminal fragments (i.e., removal of a portion of the C-terminus of a protein), C-terminal fragments (i.e., removal of a portion of the N-terminus of a protein), or internal fragments (i.e., removal of a portion of the internal part of a protein).

[0121] In the context of two polynucleotide or polypeptide sequences, “sequence identity” or “identity” refers to residues in the two sequences that are identical when aligned with respect to the greatest correspondence across a given comparison window. When the percentage of sequence identity is used in reference to a protein, non-identical residue positions are often differed by conserved amino acid substitutions, where an amino acid residue is substituted with another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity, but not limited to these) and therefore does not alter the functional properties of the molecule. If sequences differ by conserved substitutions, the percentage of sequence identity may be adjusted up to compensate for the conserved nature of the substitutions. Sequences that differ by such conserved substitutions are said to have “sequence similarity” or “similarity.” Means for making this adjustment are well known. Typically, this involves scoring the conserved substitutions as partial rather than complete mismatches, thereby increasing the percentage of sequence identity. Thus, in a non-limiting example, if identical amino acids are given a score of 1 and non-conservative substitutions are given a score of 0, then conservative substitutions may be given a score between 0 and 1. The scoring of conservative substitutions is calculated, for example, as implemented in the program PC / GENE (Intelligenetics, Mountain View, California).

[0122] The "sequence identity percentage" includes a value determined by comparing two optimally aligned sequences (the maximum number of perfectly matching residues) across a comparison window, where portions of the polynucleotide sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (which contains neither additions nor deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where identical nucleic acid bases or amino acid residues occur in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions within the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage. Unless otherwise specified (for example, if the shorter sequence contains a concatenated heterologous sequence), the comparison window is the total length of the shorter of the two sequences being compared.

[0123] Unless otherwise specified, sequence identity / similarity values ​​include values ​​obtained using GAP version 10 with the following parameters: GAP weight 50 and length weight 3 for nucleotide sequences, and identity % and similarity % using the nwsgapdna.cmp scoring matrix; GAP weight 8 and length weight 2 for amino acid sequences, and identity % and similarity % using the BLOSUM62 scoring matrix; or any equivalent program. "Equivalent program" includes any sequence comparison program that, for any two sequences in question, produces alignments having identical nucleotide or amino acid residue matches and identical sequence identity percentages compared to the corresponding alignments produced by GAP version 10.

[0124] The term "in vitro" includes artificial environments and processes or reactions occurring within them (e.g., test tubes or isolated cells or cell lines). The term "in vivo" includes natural environments (e.g., living organisms or bodies, or cells or tissues within living organisms or bodies, but not limited to these) and processes or reactions occurring within them. The term "ex vivo" includes cells removed from the body of an individual and processes or reactions occurring within such cells.

[0125] "As needed" or "as required" means that the event or situation described below may or may not occur, and that the description includes both the case in which the event or situation occurs and the case in which it does not occur.

[0126] The notation for a range of values ​​includes all integers within or defining the range, as well as all subranges defined by integers within that range.

[0127] Unless otherwise clarified by the context, the term "approximately" encompasses values ​​within ±5 of the specified value.

[0128] The term "and / or" encompasses any possible combination of one or more of the related enumerated items, as well as the absence of any combination when interpreted in the alternative form ("or").

[0129] The term "or" refers to any one member of a given list.

[0130] The singular articles "a," "an," and "the" include plural references unless otherwise explicitly specified by the context. For example, the terms "one protein" or "at least one protein" may include multiple proteins, or mixtures thereof.

[0131] Statistically significant means p ≤ 0.05. Detailed explanation I. Overview

[0132] Immunotherapy holds great promise for numerous human diseases. One of the oldest examples is bone marrow transplantation, in which the recipient's entire immune system can be replaced with autologous or allogeneic bone marrow transplantation. This procedure also allows for the correction of congenital hematopoietic failure and the regrowth of the immune system after treatment to eradicate hematological malignancies. More recent examples include immune cells engineered with antigen receptors to target tumors (e.g., CAR-T, eTCR, CAR-NK, and CAR macrophages). In all these cases, the patient must undergo a “conditioning” regimen before cell transplantation, which may function to “make a place” in the host immune niche to assist in the uptake of donor cells and, in some cases, to suppress the host-versus-graft immune response that could lead to graft rejection.

[0133] Conditioning regimens range in intensity from partial to complete myeloablative, the latter being necessary when pathogenic host immune cells must be completely eradicated (e.g., in the case of hematological malignancies). In any case, current standard treatments for host conditioning have significant drawbacks. First, conditioning agents are toxins (e.g., DNA damaging agents) that are not specific to the desired target cells, and therefore pose harmful and even life-threatening risks to the patient. Furthermore, conditioning agents are as toxic to donor cells as they are to the host, and therefore must be discontinued before transplantation so as not to interfere with life-saving cell therapy. Due to these difficulties, the application of cell therapy is usually limited to urgent cases where no other treatment options remain.

[0134] Lymphocyte suppressants have numerous potential applications for host conditioning for transplantation and adoptive cell therapy: (1) preventing allogeneic transplant rejection through T and NK cell suppression (e.g., bone marrow transplantation, gene-corrected cell therapy), (2) non-genotoxic clearance of the immune niche space for engineered cell therapy (e.g., CAR-T, TCR-T, Treg, NK, B cells, progenitor cells), (3) eliminating endogenous cytokine "syncs" and making essential factors more available to grafter cells, and (4) post-transplant immunosuppression that is less severe and less toxic than standard therapeutic agents.

[0135] A challenge to using lymphocyte suppressants as conditioning therapies is that transplanted cells, in addition to target host cells, are susceptible to their effects. Strategies to address these challenges are provided herein, including (1) the development of targeted conditioning regimens utilizing antibodies that specifically target desired host cells, for example, as monotherapy, combination therapy, bispecific antibodies, antibody-drug conjugates (ADCs), or scFv-modified CAR-Ts, and (2) the modification of donor cells to make them resistant to conditioning agents based on these antibodies. Collectively, this constitutes the concept of antibody-resistant modified receptors (ARMoRs). The underlying idea is to make minimal changes to the immune cell receptor in transplanted donor cells to disable binding by suppressive antibody agents. A schematic diagram of this strategy is shown in Figures 1A and 1B, using an example of a modified CXCR4 variant that is resistant to anti-CXCR4-mediated blockade of the interaction between CXC-motif chemokine receptor 4 (CXCR4) and its ligand CXC-motif chemokine 12 (CXCL12). The objective is to introduce a fully functional form of the receptor, unrecognized by conditioning agents, into the cell therapy product. This can be achieved by modifying the antibody recognition site, which disables binding but preserves receptor function. As a result, host cells remain sensitive to the conditioning agent, while the manipulated transplanted cells become resistant and thus gain a competitive advantage in host regrowth. The fundamental goal is to give transplanted cell therapy a competitive advantage in the host patient by applying selective pressure that specifically targets host cells while preserving donor-derived cell therapy.

[0136] A method is provided for improving the engraftment of donor cells in a subject. Such a method may include the steps of: providing donor cells expressing (e.g., modified to express) a first isoform of a target protein (e.g., CXCR4); administering the donor cells to a subject; and then selectively depleting host cells in the subject based on the expression of a second isoform of the target protein, thereby improving the engraftment of the donor cells in the subject. For example, such a method may include the steps of: providing donor cells expressing (e.g., modified to express) a first isoform of a target protein (e.g., CXCR4); administering the donor cells to a subject; and then selectively inhibiting host cells in the subject based on the expression of a second isoform of the target protein, thereby improving the engraftment of the donor cells in the subject. For example, selective inhibition may include selectively depleting host cells from the bone marrow. Alternatively, such a method may include the steps of providing donor cells expressing (e.g., modified to express) a first isoform of a target protein (e.g., CXCR4), administering the donor cells to a subject, and then selectively ablating host cells in the subject based on the expression of a second isoform of the target protein, thereby improving the engraftment of the donor cells in the subject. The donor cells may express only the first isoform, or they may express both the first and second isoforms of the target protein. The first isoform may be functionally indistinguishable from the second isoform of the target protein, but immunologically distinguishable. In some embodiments, selective inhibition of host cells may be based on the expression of only the second isoform of the target protein, and the absence of expression of the first isoform of the target protein. Alternatively, selective inhibition of host cells may be based on the expression of the second isoform, regardless of the expression of the first isoform of the target protein. Selective inhibition of host cells does not involve ablation (i.e., killing) of host cells through exogenous mechanisms, such as active killing mechanisms.Selective inhibition of host cells does not include ablation (i.e., killing) of host cells by active killing mechanisms. Active killing mechanisms mean that a drug directly kills host cells by a cytotoxic mechanism (e.g., antibody-drug conjugate (ADC), antibody-radioactive conjugate (ARC), CAR-T, or other manipulated cytotoxicity) or mobilizes host cytotoxic effector mechanisms (e.g., complement-dependent cytotoxicity (CDC), antibody-dependent cytotoxicity (ADCC), antibody-dependent cytotoxicity (ADCP)) as opposed to blocking cellular functions that do not involve exogenous cytotoxic effectors (e.g., growth or cytokine signaling, chemochemic tissue homing, cell-cell adhesion, e.g., by selective inhibition), or recruits host cytotoxic effector mechanisms (e.g., complement-dependent cytotoxicity (CDC), antibody-dependent cytotoxicity (ADCC), antibody-dependent cytophagocytosis (ADCP)). Such host cytotoxic effector mechanisms are well known. For example, see Yu et al. (2020) J. Hematol. Oncol. 13(1):45 and Gogesch et al. (2021) Int. J. Mol. Sci. 22(16):8947, each of which is incorporated herein by reference in its entirety for all purposes. As a novel conditioning strategy, selective inhibition of host cells without cytotoxic ablation has the potential to improve the safety and efficacy of cell therapy and transplantation procedures. Non-ablation conditioning can avoid the undesirable and adverse effects of ablation agents, including the direct killing of non-target (e.g., non-hematopoietic) cells expressing drug-targeted antigens, indirect toxicity to tissues adjacent to the target, and prolonged immunosuppression during the post-transplant period. Selective blockade or suppression of essential host cell factors can enhance the expansion, persistence, and transport of resistant donor cells without the use of unpleasant and potentially toxic ablation agents, by introducing desirable competition for limited host factors (e.g., cytokines, chemokines) and immune niche spaces.In some embodiments, selective inhibition of host cells may include (1) blocking the growth of host cells to provide a competitive growth advantage to donor cells, (2) blocking the localization or transport of host cells to provide a competitive homing advantage to donor cells, (3) blocking cell-cell interactions or adhesion of host cells to provide a competitive tissue invasion advantage to donor cells, or (4) blocking immune cell activation in host cells to provide a competitive advantage to donor cells. In some embodiments, selective inhibition of host cells may include selectively depleting host cells from the bone marrow. Also provided are combinations for administration to subjects requiring such combinations, comprising (1) a population of donor cells expressing (e.g., modified to express) a first isoform of a target protein (e.g., CXCR4), and (2) a drug (e.g., an antagonist, e.g., an antigen-binding protein) that specifically binds to a second isoform of the target protein but not specifically to the first isoform of the target protein.

[0137] Methods are provided for the selective depletion of unedited cells in vivo (and the regrowth of edited cells) in a subject thereof. In some embodiments, selective depletion means the selective recruitment of unedited cells from the bone marrow to the periphery. Such a method may include the steps of providing cells edited to express a first isoform of a target protein (e.g., CXC motif chemokine receptor 4 (CXCR4)), administering the edited cells to a subject, and then selectively depleting unedited cells in the subject based on the expression of a second isoform of the target protein (e.g., based on the expression of only the second isoform of the target protein and the absence of expression of the first isoform of the target protein, or based on the expression of the second isoform regardless of their expression). The first isoform may be functionally indistinguishable from the second isoform of the target protein, but immunologically distinguishable. Edited cells may express only the first isoform, or they may express both the first and second isoforms of the target protein. Also provided are combinations for administration to subjects requiring such combinations, comprising (1) a population of cells edited to express a first isoform of a target protein (e.g., CXCR4), and (2) a drug (e.g., an antagonist, e.g., an anti-CXCR4 antigen-binding protein) that specifically binds to a second isoform of the target protein but not to the first isoform of the target protein.

[0138] Also provided are isolated cells or populations of cells modified to express a first isoform of a target protein (e.g., CXCR4) that is different from a second isoform of the target protein. The cells may express only the first isoform, or they may express both the first and second isoforms. The first isoform may be functionally indistinguishable from the second isoform, but immunologically distinguishable. The cells may express only the first isoform, or they may express both the first and second isoforms. Also provided are isolated cells or populations of cells in which a genomic locus has been edited to express a first isoform of a target protein that is different from a second isoform. Methods for producing such cells are also provided.

[0139] Also provided are isolated cells or populations of cells edited (i.e., modified) to express a first isoform of CXCR4 distinct from a second isoform of CXCR4. The first isoform may be functionally indistinguishable from the second isoform of the target protein, but immunologically distinguishable. The edited cells may express only the first isoform, or they may express both the first and second isoforms of the target protein. Methods for producing such cells are also provided, as are the engineered CXCR4 protein and the nucleic acids encoding the engineered CXCR4 protein.

[0140] In some embodiments, the cells (e.g., donor cells or edited cells) in the compositions and methods contain or express therapeutic molecules, such as therapeutic proteins or enzymes, immunoglobulins (e.g., antibodies or their antigen-binding fragments), chimeric antigen receptors (CARs) (e.g., CAR-T cells, CAR-NK cells), or exogenous T cell receptors (TCRs). In some embodiments, the therapeutic molecules, immunoglobulins, CARs, or exogenous TCRs do not target a target protein (e.g., CXCR4). For example, donor cells or edited cells may be engineered to express therapeutic molecules that have therapeutic activity against any disease, such as any type of cancer (e.g., regardless of whether the target protein is associated with the disease or cancer), including diseases or cancers unrelated to the target protein (for example, the target proteins discussed above are not targeted to treat diseases or cancers, but the compositions and methods disclosed herein may offer a competitive advantage to cells containing or expressing therapeutic molecules). For example, the disease or cancer may be unrelated to the target protein (e.g., the target protein does not cause either the disease or the cancer, and / or the expression of the target protein is unrelated to either the disease or the cancer). In some such embodiments, the therapeutic molecule may target affected cells and / or antigens expressed on affected cells (e.g., tumor-associated antigens). II. Methods to improve donor cell engraftment in subjects

[0141] In some embodiments of the present invention, a method is provided for improving the engraftment of donor cells in a subject. Such a method may include the step of providing donor cells that express (e.g., are modified to express) a first isoform of a target protein (e.g., CXCR4) different from a second isoform of the target protein, wherein the second isoform is expressed in host cells of the subject. The target protein may be, for example, CXCR4. The first isoform may be functionally indistinguishable from the second isoform of the target protein, but immunologically distinguishable. In some embodiments, the donor cells express only the first isoform of the target protein. In other embodiments, the donor cells express both the first and second isoforms of the target protein. Such a method may include the step of providing donor cells in which a target genomic locus has been edited to express the first isoform of the target protein. The donor cells may then be administered to a subject, selectively depleting host cells in the subject based on the expression of the second isoform of the target protein, thereby improving the engraftment of the donor cells in the subject. Similarly, donor cells can then be administered to a subject, providing the subject with a means to specifically bind to the second isoform of CXCR4 but not specifically to the first isoform of CXCR4. For example, selective depletion of host cells may be based on the expression of only the second isoform of the target protein and the absence of expression of the first isoform of the target protein. Alternatively, selective depletion of host cells may be based on the expression of the second isoform of the target protein, regardless of the expression of the first isoform of the target protein. For example, host cells in a subject can be selectively inhibited based on the expression of the second isoform of the target protein, thereby improving the engraftment of donor cells in the subject. For example, selective inhibition of host cells may be based on the expression of only the second isoform of the target protein and the absence of expression of the first isoform of the target protein. Alternatively, selective inhibition of host cells may be based on the expression of the second isoform of the target protein, regardless of the expression of the first isoform of the target protein.Alternatively, host cells in a target can be selectively ablated based on the expression of a second isoform of the target protein, thereby improving the engraftment of donor cells in the target. For example, selective ablation of host cells may be based on the expression of only the second isoform of the target protein and the absence of expression of the first isoform of the target protein. Alternatively, selective depletion of host cells may be based on the expression of the second isoform, regardless of the expression of the first isoform of the target protein. In some embodiments, selective inhibition of host cells includes selectively depleting host cells from the bone marrow.

[0142] In some embodiments of the present invention, a method is provided for the selective depletion of unedited cells (and the regrowth of edited cells) in a target. In some embodiments, selective depletion means the selective recruitment of unedited cells from the bone marrow to the periphery. Such a method may include the step of providing edited cells that have been modified to express a first isoform of a target protein different from a second isoform of the target protein, wherein the second isoform is expressed in the unedited cells of the target. The first isoform may be functionally indistinguishable from the second isoform of the target protein, but may be immunologically distinguishable. In some embodiments, the edited cells express only the first isoform of the target protein (e.g., CXCR4). In other embodiments, the edited cells express both the first and second isoforms of the target protein (e.g., CXCR4). Edited cells can then be administered to a target, selectively depleting unedited cells in the target based on the expression of a second isoform of the target protein (e.g., based on the expression of only the second isoform of the target protein and the absence of the expression of the first isoform of the target protein, or based on the expression of the second isoform regardless of the expression of the first isoform of the target protein). Such selective depletion may, in non-limited cases, involve bone marrow egress of unedited cells in the target to "make a place" for the engraftment of edited cells administered to the target in the host immune niche.

[0143] Donor cells or edited cells may be any suitable cells. Similarly, host cells or unedited cells may be any suitable cells. In some embodiments, the cells are immune cells. In some embodiments, the cells are hematopoietic cells. The term hematopoietic cells refers to cells originating from hematopoietic stem cells or hematopoietic progenitor cells, and / or from erythroblast, lymphoid, or myeloid lineages. In some embodiments, the cells are immune cells. The term immune cells refers to any cells derived from hematopoietic stem cells that play a role in the immune response. Immune cells include, but are not limited to, lymphocytes, e.g., T cells and B cells, antigen-presenting cells (APCs), dendritic cells, monocytes, macrophages, natural killer (NK) cells, mast cells, basophils, eosinophils, or neutrophils, and any precursors of such cells. In some embodiments, the cells are lymphocytes or lymphoid progenitor cells. In some embodiments, the cells are T cells (e.g., CD4+ T cells, CD8+ T cells, memory T cells, regulatory T cells, gamma delta T cells, mucosa-associated invariant T cells (MAITs), tumor-infiltrating lymphocytes (TILs), or any combination thereof). In some embodiments, the cells are alpha-beta T cells. In some embodiments, the cells are gamma delta T cells. In some embodiments, the cells are TILs. In some embodiments, the cells are B cells. In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are innate lymphoid cells. In some embodiments, the cells are dendritic cells. In some embodiments, the cells are hematopoietic stem cells (HSCs) or hematopoietic stem and progenitor cells (HSPCs) or their offspring. HSCs can give rise to both myeloid and lymphoid progenitor cells, which further give rise to myeloid cells (e.g., monocytes, macrophages, neutrophils, basophils, dendritic cells, erythrocytes, platelets, etc.) and lymphoid cells (e.g., T cells, B cells, NK cells), respectively. In some embodiments, the cells are derived from induced pluripotent stem cells (e.g., NK cells derived from induced pluripotent stem cells). In some embodiments, the cells are derived from HSCs or HSPCs.

[0144] In some embodiments, cells (e.g., donor cells or edited cells) include genetic modifications that correct or counteract disease-related gene deficiencies present in the subject (e.g., insertion of a transgene, correction of a mutation, deletion or inactivation of a gene (e.g., insertion of an immature stop codon or insertion of a regulatory repressor sequence), or changes in epigenetic modifications that are important for gene expression). In some embodiments, cells (e.g., donor cells or edited cells) include a transgene. In some embodiments, cells (e.g., donor cells or edited cells) include or express therapeutic molecules, e.g., therapeutic proteins or enzymes, immunoglobulins (e.g., antibodies or their antigen-binding fragments), chimeric antigen receptors (CARs) (e.g., CAR-T cells, CAR-NK cells), or exogenous T cell receptors (TCRs). In some embodiments, cells (e.g., donor cells or edited cells) include a bicistronic nucleic acid construct encoding a therapeutic molecule and a first isoform of the target protein. See, for example, Yeku et al. (2017) Sci. Rep. 7(1):10541 and Rafiq et al. (2018) Nat. Biotechnol. 36(9):847-856, each of which, for any purpose relating to bicistronic constructs for expressing, for example, CAR and another molecule, are incorporated herein by reference in their entirety. For example, a bicistronic construct may encode both a therapeutic protein (e.g., CAR, TCR or antigen-binding fragment of TCR, or immunoglobulin) and a first isoform (e.g., a modified isoform) of a target protein (e.g., CXCR4). In one embodiment, the bicistronic construct encodes a therapeutic protein (e.g., CAR, TCR or antigen-binding fragment of TCR, or immunoglobulin) and a modified isoform of CXCR4. In some embodiments, the therapeutic molecule, immunoglobulin, CAR, or exogenous TCR does not target a target protein (e.g., CXCR4). In some embodiments, the cells (e.g., donor cells or edited cells) contain or express immunoglobulins, CARs, or exogenous TCRs.In some embodiments, cells (e.g., donor cells or edited cells) contain or express CARs or exogenous TCRs. For example, donor cells or edited cells may be engineered to express a therapeutic molecule having therapeutic activity against any disease, e.g., any type of cancer (e.g., regardless of whether the target protein is associated with the disease or cancer), including diseases or cancers unrelated to the target protein (e.g., the target proteins discussed above are not targeted to treat diseases or cancers, but the compositions and methods disclosed herein may offer a competitive advantage to cells containing or expressing the therapeutic molecule). In some embodiments, the therapeutic molecule targets affected cells and / or antigens expressed on affected cells (e.g., tumor-associated antigens). For example, the disease or cancer may be a disease or cancer unrelated to the target protein (e.g., the target protein does not cause either the disease or the cancer, and / or the expression of the target protein is unrelated to either the disease or the cancer). Exemplary types of cancers and tumors that can be treated are described elsewhere herein.

[0145] In some embodiments, the donor cells are autologous (i.e., derived from the subject). In some embodiments, the donor cells are homogeneous (i.e., not derived from the subject) or syngeneic (i.e., genetically identical or sufficiently identical, immunologically compatible, and acceptable for transplantation). In some embodiments, the cells are mammalian cells or non-human mammalian cells (e.g., mouse or rat cells or non-human primate cells) (e.g., the subject is a mammal or non-human mammal, and the donor cells are mammalian cells or non-human mammalian cells). In some embodiments, the donor cells are human cells (e.g., the subject is human, and the cells are human cells).

[0146] In some embodiments of the present invention, the target protein is CXC chemokine receptor 4 (also known as CXC-motif chemokine receptor 4, CXCR4, FB22, Fusin, HM89, LCR1, leukocyte-derived 7-transmembrane domain receptor (LESTR), lipopolysaccharide-related protein 3 (LAP-3, LPS-related protein 3), NPY3R, NPYRL, stromal cell-derived factor 1 receptor (SDF-1 receptor), and CD184). CXCR4 is a 7-transmembrane receptor for the CXC chemokine CXCL12 / SDF-1, which plays a role in cell migration and signal transduction by increasing intracellular calcium ion levels and enhancing MAPK1 / MAPK3 activation. CXCR4 can also act as a receptor for extracellular ubiquitin, and its binding results in increased intracellular calcium levels and decreased cellular cAMP. CXCR4 is involved in hematopoiesis, ventricular septal formation, and plays an essential role in gastrointestinal angiogenesis. CXCR4 also plays several roles in the pathogen response, including binding to bacterial lipopolysaccharide (LPS) and mediating LPS-induced inflammatory responses, including TNF secretion by monocytes, and acting as a co-receptor (with CD4) for human immunodeficiency virus (HIV) to promote Env-mediated fusion between the virus and T cells.

[0147] In some embodiments, the target protein is human CXCR4. Human CXCR4 is assigned UniProt accession number P61073. The canonical isoform of human CXCR4 is assigned UniProt accession number P61073-1 and NCBI accession number NP_003458.1 and is described in Sequence ID 1. An exemplary messenger RNA encoding the canonical isoform of human CXCR4 is assigned NCBI accession number NM_003467.3 and is described in Sequence ID 2. The coding sequence for the canonical isoform of human CXCR4 is assigned CCDS ID CCDS46420.1 and is described in Sequence ID 3. The gene encoding human CXC motif chemokine receptor 4 is called CXCR4, is located on chromosome 2, and is assigned NCBI GeneID 7852. This is located at position 2q22.1 (assembly:GRCh38.p14(GCF_000001405.40), position:NC_000002.12(136114349..136118149, complementary)).

[0148] The expression "functionally indistinguishable" refers to first and second isoforms that can perform the same function (e.g., binding to an endogenous ligand (e.g., CXCL12 for CXCR4) and / or activation of a downstream signaling pathway (e.g., the CXCR4 signaling pathway)) equally within the cell (e.g., without significant impairment). In other words, the first and second isoforms are functionally largely indistinguishable. In certain embodiments, slight functional impairment is acceptable. The function that is largely indistinguishable may be, for example, binding to an endogenous ligand (e.g., CXCL12 for CXCR4) and / or activation of a downstream signaling pathway. In some embodiments, the function may be binding to an endogenous ligand (e.g., CXCL12 for CXCR4) and activation of a downstream signaling pathway. The expression "immunologically distinct" means that the first and second isoforms of a protein can be distinguished by an antigen-binding protein (e.g., one that specifically binds to either the first or second isoform but not to the other), such as an antigen-binding protein that specifically binds only to the second (unmodified) isoform of the target protein. In other words, an antigen-binding protein can distinguish between two isoforms by specifically binding to only one isoform but not to the other. In certain embodiments, an endogenous ligand (e.g., CXCL12 for CXCR4) binds to both the first and second isoforms (e.g., equally or with only slight impairment), while a manipulated antigen-binding protein, such as an antibody, can distinguish between the two isoforms by specifically binding to only one isoform but not to the other (e.g., specifically binding only to the second isoform but not to the first isoform).

[0149] In some embodiments, the second isoform of the target protein refers to the form present in the subject. In some embodiments, the second isoform of the target protein refers to the wild-type or native form of the target protein (i.e., the form normally found in nature), and the first isoform refers to the isoform obtained by introducing a mutation into the nucleic acid sequence encoding the second isoform. The native form of a protein refers to the protein encoded by the nucleic acid sequence in the cell's genome, without any insertions or mutations by genetic manipulation (i.e., a native protein is a protein that is neither a transgenic protein nor a genetically modified protein).

[0150] Mutations in the first isoform can be of any type and of any size. In some embodiments, a mutation involves the insertion, deletion, and / or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids (e.g., 1 to 20 amino acids, 1 to 5 amino acids, 1 to 3 amino acids, or 1 amino acid). In some embodiments, a mutation involves one or more (e.g., 3) substitutions (e.g., one or more (e.g., 3) substitutions of each amino acid). In some embodiments, a mutation consists essentially of one or more (e.g., 3) substitutions (e.g., one or more (e.g., 3) substitutions of each amino acid). In some embodiments, the mutation consists of one or more (e.g., three) substitutions (e.g., one or more (e.g., three) substitutions of one amino acid each). In some embodiments, the mutation includes an insertion (e.g., an insertion of one amino acid). In some embodiments, the mutation is essentially an insertion (e.g., essentially an insertion of one amino acid). In some embodiments, the mutation consists of an insertion (e.g., an insertion of one amino acid). The mutation can be at any site in the target protein. For example, if the target protein is a cell surface protein, the mutation may, in some embodiments, be in the extracellular domain of the target protein. In some embodiments, the site of the mutation may be a site that is non-conserved across different mammalian species. In some embodiments, the mutation does not result in a change in the secondary structure of the surface protein. In some embodiments, the mutation is located within an epitope targeted by an antigen-binding protein or at a site accessible for ligand binding. In some embodiments, the mutation is not located at a site in the surface protein that is involved in a predicted or experimentally established or confirmed protein-protein interaction. In some embodiments, the mutation does not result in the deletion or introduction of disulfide bonds, intermolecular or intramolecular interactions, or hydrophobic stacking.In some embodiments, the mutation does not result in the deletion or introduction of a post-translational protein modification site, such as a glycosylation site. In some embodiments, the mutation is located in a site that has a unique topology compared to other mammalian proteins, as determined by crystal structure analysis or computer-aided structural prediction. In cases where an antibody or antigen-binding protein that is reactive to the target protein already exists, the site of the mutation can be selected using information about the epitope of the target protein recognized by that antibody or antigen-binding protein.

[0151] In some embodiments, the first isoform of the target protein is a genetically engineered isoform of the target protein. For example, the first isoform of the target protein may be genetically engineered to include a mutation (e.g., an artificial mutation not naturally occurring) that provides a modified epitope. The modified epitope may be, for example, located in the binding region of an antigen-binding protein such as an antibody. In some embodiments, the target protein is CXCR4 (e.g., human CXCR4), and the modified epitope is located in the binding region of a REGN7663 or REGN7664 anti-CXCR4 antibody, as described elsewhere herein. The ECL2 region of human CXCR4 (SEQ ID NO: 1), at positions A175-V198, is necessary and sufficient for the binding of REGN7663 and REGN7664. In some embodiments, the mutation may include a mutation (e.g., substitution and / or insertion) encoded by a nucleotide in coding exon 2 of the CXCR4 gene (e.g., the human CXCR4 gene). In some embodiments, the mutation may include a mutation (e.g., substitution and / or insertion) within the ECL2 region of CXCR4 (e.g., human CXCR4). In some embodiments, the mutation may include a mutation (e.g., substitution and / or insertion) within the region A175 to V198 of CXCR4 (e.g., human CXCR4 or the corresponding region of CXCR4 when aligned with human CXCR4). In some embodiments, the mutation may include a mutation (e.g., substitution and / or insertion) within the region S178 to R183 of CXCR4 (e.g., human CXCR4 or the corresponding region of CXCR4 when aligned with human CXCR4). In some embodiments, the mutation may include a mutation (e.g., substitution and / or insertion) within the region R188 to L194 of CXCR4 (e.g., human CXCR4 or the corresponding region of CXCR4 when aligned with human CXCR4). In some embodiments, the mutation may include mutations (e.g., substitutions and / or insertions) within the REGN7663 and REGN7664 binding region (SEQ ID NO: 57).In some embodiments, the mutations may include mutations (e.g., substitutions and / or insertions) at one or more of the following locations in CXCR4 (e.g., human CXCR4 or the corresponding region of CXCR4 when aligned with human CXCR4): A175, N176, V177, S178, E179, A180, D181, D182, R183, Y184, I185, C186, D187, R188, F189, Y190, P191, N192, D193, L194, W195, V196, V197, and V198. In some embodiments, the mutation may include a mutation (e.g., substitution) at one or more of the following locations in CXCR4 (e.g., human CXCR4 or the corresponding region of CXCR4 when aligned with human CXCR4): A175, N176, V177, S178, E179, A180, D181, D182, R183, Y184, I185, C186, D187, R188, F189, Y190, P191, N192, D193, L194, W195, V196, V197, and V198. In some embodiments, the mutation occurs between A175 and N176, between N176 and V177, between V177 and S178, between S178 and E179, between E179 and A180, between A180 and D181, between D181 and D182, and between D182 and R183 in CXCR4 (e.g., human CXCR4 or the corresponding region of CXCR4 when aligned with human CXCR4). The mutations may include those between R183 and Y184, between Y184 and I185, between I185 and C186, between C186 and D187, between D187 and R188, between R188 and F189, between F189 and Y190, between Y190 and P191, between P191 and N192, between N192 and D193, and / or between D193 and L194 (e.g., insertions). In some embodiments, the mutations may include those between S178 and E179 (e.g., insertions).In some embodiments, mutations may include mutations (e.g., substitutions) in one or more of the following locations in CXCR4 (e.g., human CXCR4 or the corresponding region of CXCR4 when aligned with human CXCR4): A175, N176, V177, S178, E179, A180, D181, D182, R183, Y184, I185, C186, D187, R188, F189, Y190, P191, N192, D193, L194, W195, V196, V197, and V198, and mutations (e.g., insertions) between S178 and E179. In some embodiments, mutations may include the mutations (e.g., substitutions and / or insertions) or combinations of mutations listed in Table 1.

[0152] [Table 1-1] [Table 1-2]

[0153] In some embodiments, mutations may include mutations (e.g., substitutions and / or insertions) or combinations of mutations described in pMM626, pMM630, pMM632, pMM633, and pMM640 (corresponding to sequence numbers 91, 95, 97, 98, and 105, respectively). For example, mutations may include mutations (e.g., substitutions) at positions F189, N192, D193, E179, D181, and / or D182 of CXCR4 (e.g., human CXCR4 or the corresponding region of CXCR4 when aligned with human CXCR4), mutations (e.g., insertions) between S178 and E179 of CXCR4 (e.g., human CXCR4 or the corresponding region of CXCR4 when aligned with human CXCR4), or any combination thereof of CXCR4 (e.g., human CXCR4). Mutations at a location within CXCR4 include mutations that involve only the residue at that location (e.g., substitutions and / or insertions), or mutations that involve the residue at that location as well as other residues at other locations (e.g., substitutions and / or insertions). The amino acid position nomenclature for mutations or residues disclosed herein refers to the location of the mutation or residue in the canonical isoform of human CXCR4 described in Sequence ID No. 1.

[0154] In some embodiments, the mutation includes a mutation (e.g., substitution) at position F189 of CXCR4 (e.g., human CXCR4 or the corresponding region of CXCR4 when aligned with human CXCR4). A preferred example of an F189 substitution is the F189A substitution. In some embodiments, the mutation includes a mutation (e.g., substitution) at position N192 of CXCR4 (e.g., human CXCR4 or the corresponding region of CXCR4 when aligned with human CXCR4). A preferred example of an N192 substitution is the N192A substitution. In some embodiments, the mutation includes a mutation (e.g., substitution) at position D193 of CXCR4 (e.g., human CXCR4 or the corresponding region of CXCR4 when aligned with human CXCR4). A preferred example of a D193 substitution is the D193A substitution. In some embodiments, the mutations include mutations (e.g., substitutions) at positions F189, N192, and D193 of CXCR4 (e.g., human CXCR4 or the corresponding region of CXCR4 when aligned with human CXCR4). Preferred examples of F189, N192, and D192 substitutions are F189A, N192A, and D192A substitutions. In some embodiments, the mutations include insertions between S178 and E179 of CXCR4 (e.g., human CXCR4 or the corresponding region of CXCR4 when aligned with human CXCR4). Preferred examples of insertions include S178_E179insK (e.g., insertion of K between S178 and E179), S178_E179insY (e.g., insertion of Y between S178 and E179), and S178_E179insR (e.g., insertion of R between S178 and E179). In some embodiments, the mutation includes a mutation (e.g., substitution) at position E179 of CXCR4 (e.g., human CXCR4 or the corresponding region of CXCR4 when aligned with human CXCR4). A preferred example of an E179 substitution is the E179R substitution. In some embodiments, the mutation includes a mutation (e.g., substitution) at position D181 of CXCR4 (e.g., human CXCR4 or the corresponding region of CXCR4 when aligned with human CXCR4). A preferred example of a D181 substitution is the D181R substitution.In some embodiments, the mutation includes a mutation (e.g., substitution) at position D182 of CXCR4 (e.g., human CXCR4 or the corresponding region of CXCR4 when aligned with human CXCR4). A preferred example of a D182 substitution is the D182R substitution. In some embodiments, the mutation includes mutations (e.g., substitutions) at positions E179, D181, and D182 of CXCR4 (e.g., human CXCR4 or the corresponding region of CXCR4 when aligned with human CXCR4). Preferred examples of E179, D181, and D182 substitutions are the E179R, D181R, and D182R substitutions.

[0155] In some embodiments, the mutation includes the mutation (e.g., substitution) described in SEQ ID NO: 91 for SEQ ID NO: 57 or for positions 175-198 of SEQ ID NO: 1. In other words, in some embodiments, the mutation includes, is essentially, or consists of F189A, N192A, and N193A for SEQ ID NO: 1 (or the corresponding region of CXCR4 when aligned with human CXCR4).

[0156] In some embodiments, the mutation includes the mutation (e.g., insertion) described in SEQ ID NO: 95 for SEQ ID NO: 57, or for positions 175-198 of SEQ ID NO: 1. In other words, in some embodiments, the mutation includes, is essentially, or consists of S178_E179insK for SEQ ID NO: 1 (or the corresponding region of CXCR4 when aligned with human CXCR4).

[0157] In some embodiments, the mutation includes the mutation (e.g., insertion) described in SEQ ID NO: 97 for SEQ ID NO: 57, or for positions 175-198 of SEQ ID NO: 1. In other words, in some embodiments, the mutation includes, is essentially, or consists of S178_E179insY for SEQ ID NO: 1 (or the corresponding region of CXCR4 when aligned with human CXCR4).

[0158] In some embodiments, the mutation includes the mutation (e.g., insertion) described in SEQ ID NO: 98 for SEQ ID NO: 57, or for positions 175-198 of SEQ ID NO: 1. In other words, in some embodiments, the mutation includes, is essentially, or consists of S178_E179insR for SEQ ID NO: 1 (or the corresponding region of CXCR4 when aligned with human CXCR4).

[0159] In some embodiments, the mutation includes the mutation (e.g., substitution) described in SEQ ID NO: 105 for SEQ ID NO: 57 or for positions 175-198 of SEQ ID NO: 1. In other words, in some embodiments, the mutation includes, is essentially, or consists of E179R, D181R, and D182R for SEQ ID NO: 1 (or the corresponding region of CXCR4 when aligned with human CXCR4).

[0160] Donor cells or edited cells may be administered to a subject by any preferred means. The term "administering" refers to the administration of a composition (e.g., donor cells or edited cells) to a subject or system (e.g., cells, organs, tissues, organisms, or their associated components or sets of components, but not limited to these). The route of administration may vary depending, for example, to the subject or system to which the composition is administered, the nature of the composition, the purpose of the administration, etc. The terms "administering" or "administering" are intended to include a route for introducing donor cells or edited cells to a subject in order to perform the intended function. Non-limiting examples of routes of administration that may be used in some embodiments include, for example, injection (subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal), such as intravenous injection. In some embodiments of the present invention, donor cells or edited cells are administered by intravenous injection. Administration may include intermittent or continuous administration (e.g., perfusion) for at least a selected period of time. Donor cells or edited cells may be administered alone or in combination with any other agent (e.g., an agent for selective inhibition or depletion of host cells or unedited cells in the target) or a pharmaceutically acceptable carrier, or both. Donor cells or edited cells may be administered before, concurrently with, or after the administration of other agents.

[0161] Host cells or unedited cells in a target can be selectively inhibited or depleted (e.g., recruited from their immune cell niche) by any suitable means based on the expression of a second isoform of the target protein (e.g., based on the expression of only the second isoform of the target protein and the absence of the expression of the first isoform of the target protein, or based on the expression of the second isoform regardless of the expression of the first isoform of the target protein). For example, they may be depleted or inhibited based on the expression of only the second isoform of the target protein and the absence of the expression of the first isoform of the target protein. Alternatively, they may be depleted or inhibited based on the expression of the second isoform regardless of the expression of the first isoform of the target protein. In some embodiments, selective inhibition or selective depletion means the selective recruitment of host cells or unedited cells from the bone marrow to the periphery. Selective inhibition or selective depletion of host cells or unedited cells may be performed before administration of donor cells or edited cells, concurrently with administration of donor cells or edited cells, or after administration of donor cells or edited cells. Selective depletion refers to the selective reduction of the total number or concentration (e.g., in the bone marrow) of cells expressing a particular isoform of a target protein. Selective depletion of cells expressing a second isoform may correspond to enrichment of cells expressing a first isoform.

[0162] In some embodiments, selective depletion refers to the selective ablation of host cells. Selective ablation of host cells refers to the ablation (i.e., killing) of host cells by an active killing mechanism. An active killing mechanism means that, in contrast to the blockade of cellular functions that do not involve exogenous cytotoxic effectors (e.g., growth or cytokine signaling, chemotactic tissue homing, cell-cell adhesion, e.g., by selective inhibition), the drug directly kills host cells by a cytotoxic mechanism (e.g., antibody-drug conjugate (ADC), antibody-radioactive conjugate (ARC), CAR-T, or other manipulated cytotoxicity) or mobilizes host cytotoxic effector mechanisms (e.g., complement-dependent cytotoxicity (CDC), antibody-dependent cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP)). In some embodiments, selective depletion of host cells or unedited cells includes ablation of host cells or unedited cells by active killing mechanisms, such as complement-dependent cell-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), antibody-drug conjugates (ADC), CAR-T, or other manipulated cytotoxicity.

[0163] In other embodiments, selective depletion refers to selective inhibition of host cells. In contrast to selective ablation, selective inhibition of host cells does not involve ablation (i.e., killing) of host cells by an active killing mechanism. That is, selective inhibition of host cells does not involve cytotoxic ablation of host cells. In some embodiments, selective inhibition of host cells or non-edited cells does not involve ablation or killing of host cells or non-edited cells, even indirectly. In some embodiments, selective inhibition or depletion of host cells or unedited cells includes (1) providing a competitive growth advantage to donor cells or edited cells by blocking the growth of host cells or unedited cells (e.g., by blocking proliferation or immune cell activation), (2) providing a competitive homing advantage to donor cells or edited cells by blocking the localization or transport of host cells or unedited cells, (3) providing a competitive tissue invasion advantage to donor cells or edited cells by blocking cell-cell interactions or adhesion of host cells or unedited cells, or (4) providing a competitive advantage to donor cells or edited cells by blocking immune cell activation in host cells or unedited cells. For example, in some embodiments, selective inhibition or depletion of host cells or unedited cells includes selectively depleting host cells from the bone marrow. As a novel conditioning strategy, selective inhibition of host cells without cytotoxic ablation has the potential to improve the safety and efficacy of cell therapies and transplantation procedures. Non-ablation conditioning can avoid the undesirable and adverse effects of ablation agents, including direct killing of non-target (e.g., non-hematopoietic) cells expressing drug-targeted antigens, indirect toxicity to tissues adjacent to the target, and prolonged immunosuppression during the post-transplant period. Selective blockade or suppression of essential host cell factors can enhance the expansion, persistence, and transport of resistant donor cells without the use of unpleasant and potentially toxic ablation agents by creating desirable competition for limited host factors (e.g., cytokines, chemokines) and immune niche spaces.

[0164] In some embodiments of the present invention, the method may include the step of providing a subject with means to specifically bind to a second isoform of CXCR4 but not to a first isoform of CXCR4. In some embodiments of the present invention, selective inhibition or selective depletion of host cells or non-edited cells in the subject may include providing a subject with means to specifically bind to a second isoform of CXCR4 but not to a first isoform of CXCR4. In some embodiments of the present invention, selective inhibition or selective depletion of host cells or non-edited cells in the subject may include administering a drug (e.g., an antagonist, an antigen-binding protein, or a population of cells expressing an antigen-binding protein (i.e., immune effector cells, e.g., chimeric antigen receptor T cells (CAR-T))) that specifically binds to a second isoform of the target protein but not to a first isoform of the target protein. For example, the drug may be an antagonist that blocks the interaction between the endogenous ligand and the second isoform of the target protein but does not block the interaction between the endogenous ligand and the first isoform of the target protein. In some embodiments of the present invention, selective inhibition or depletion of host cells or non-edited cells in a subject may include administering an antagonist (e.g., an antigen-binding protein, or a population of cells expressing an antigen-binding protein (i.e., immune effector cells, e.g., chimeric antigen receptor T cells (CAR-T))) that specifically binds to a second isoform of the target protein but not to a first isoform of the target protein (e.g., an antagonist that blocks the interaction between an endogenous ligand (e.g., CXCL12) and a second isoform of the target protein (e.g., CXCR4), but does not block the interaction between the endogenous ligand and a first isoform of the target protein).In some embodiments of the present invention, selective inhibition or depletion of host cells or unedited cells in a subject may include administering an antigen-binding protein (e.g., an isolated antigen-binding protein) or an antigen-binding protein, such as an antibody (e.g., a human antibody, a monoclonal antibody, and / or a recombinant antibody) or one or more nucleic acids encoding such an antigen-binding fragment, which specifically binds to a second isoform of the target protein (or its antigen fragment (e.g., the extracellular domain)) but not to a first isoform of the target protein. In some embodiments of the present invention, selective inhibition or depletion of host cells or unedited cells in a subject may include administering a population of cells expressing an antigen-binding protein that specifically binds to a second isoform of the target protein but not to a first isoform of the target protein (i.e., immune effector cells) (e.g., T cells expressing a chimeric antigen receptor or an exogenous T cell receptor). Immune effector cells are cells that can perform or enhance an immune response. In some embodiments (i.e., with respect to selective ablation), selective depletion (e.g., selective ablation) can be achieved by cytotoxicity mechanisms (e.g., antibody-drug conjugates (ADCs), antibody-radioactive conjugates (ARCs), CAR-T, or other manipulated cytotoxicity). In some embodiments (i.e., with respect to selective ablation), selective depletion (e.g., selective ablation) can be achieved by mobilizing host cytotoxicity effector mechanisms (e.g., complement-dependent cell-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP)) as opposed to blocking cellular functions that do not involve exogenous cytotoxicity effectors (e.g., growth or cytokine signaling, chemotactic tissue homing, cell-cell adhesion, e.g., by selective inhibition). In some embodiments (i.e., with respect to selective ablation), antigen-binding proteins are linked to toxins, thereby forming immunotoxins. In some embodiments, antigen-binding proteins are not linked to toxins.In some embodiments, the antigen-binding protein is a bispecific antigen-binding protein capable of simultaneously binding to two different antigens. In some embodiments, selective depletion (e.g., selective ablation) can be achieved by complement-dependent cytotoxicity (CDC), antibody-dependent cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), or antibody-drug conjugate (ADC). In some embodiments, selective inhibition or selective depletion is not achieved by cytotoxic mechanisms or by mobilizing host cytotoxic effector mechanisms. In some embodiments, selective inhibition or selective depletion is achieved by blocking cellular functions (e.g., growth or cytokine signaling, chemotactic tissue homing, cell-cell adhesion) without the involvement of exogenous cytotoxic effectors. For example, selective inhibition or selective depletion may be achieved by selectively depleting host cells from the bone marrow. In some embodiments, selective inhibition or depletion is not achieved by complement-dependent cell-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), or antibody-drug conjugates (ADCs). Toxins or drugs suitable for use in antibody-drug conjugates are well known in the art. See, for example, Peters et al. (2015) Biosci. Rep. 35(4):e00225, Beck et al. (2017) Nature Reviews Drug Discovery 16:315-337; Marin-Acevedo et al. (2018) J. Hematol. Oncol. 11:8; Elgundi et al. (2017) Advanced Drug Delivery Reviews 122:2-19, each of which is incorporated herein by reference in whole for all purposes. In some embodiments, the antibody-drug conjugate may further include an antigen-binding protein (e.g., an antibody) and a linker (e.g., a peptide linker, e.g., a cleavage linker) that binds the drug molecule.Selective inhibition or selective depletion can also be performed by administering antigen-binding proteins that are not linked to effector compounds such as drugs or toxins. In some embodiments, selective inhibition or selective depletion is achieved by blocking binding by endogenous ligands (e.g., CXCL12 for CXCR4).

[0165] In a method of administering a drug for selective inhibition or depletion of host cells or non-edited cells, the drug may, in some embodiments, be administered simultaneously with donor cells or edited cells. In some embodiments, the donor cells or edited cells are administered after the drug. For example, in some embodiments, the donor cells or edited cells are administered within one day after the drug, or at least about one day, at least two days, at least three days, at least four days, at least five days, at least six days, at least one week, at least two weeks, at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least eleven weeks, at least twelve weeks, at least three months, at least four months, at least five months, at least six months, or later. In some embodiments, the donor cells or edited cells are administered before the drug. For example, in some embodiments, donor cells or edited cells are administered within one day prior to the drug, or at least approximately one day, at least two days, at least three days, at least four days, at least five days, at least six days, at least one week, at least two weeks, at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least eleven weeks, at least twelve weeks, at least three months, at least four months, at least five months, at least six months, or earlier.

[0166] In some embodiments, donor cells or edited cells are administered in multiple doses (e.g., doses). In some embodiments, donor cells or edited cells are administered to the subject once. In some embodiments, donor cells or edited cells are administered to the subject more than once (e.g., at least two, at least three, at least four, at least five, or more times). In some embodiments, donor cells or edited cells are administered to the subject at regular intervals (e.g., every six months). In a method of administering a drug for selective inhibition or depletion of host cells or non-edited cells, it may be administered in multiple doses (e.g., doses) in some embodiments. In some embodiments, the drug is administered to the subject once. In some embodiments, the drug is administered to the subject more than once (e.g., at least two, at least three, at least four, at least five, or more times). In some embodiments, the drug is administered to the subject at regular intervals (e.g., every six months).

[0167] In some embodiments, the drug is administered to the subject before and after the administration of donor cells or edited cells. In some embodiments, the drug is administered to the subject before and after the administration of donor cells or edited cells, and the donor cells or edited cells are administered to the subject once. In some embodiments, for example, the drug is administered to the subject about 1 to 2 weeks before the administration of donor cells or edited cells, and about 1 to 2 weeks after the administration of donor cells or edited cells (for example, to provide a competitive advantage to the donor cells or edited cells).

[0168] In some embodiments, the agent is administered to the subject in multiple doses (e.g., at least 2 doses, at least 3 doses, at least 4 doses, at least 5 doses, or more doses) before administration of the donor cells or edited cells. In some embodiments, the agent is administered to the subject in multiple doses (e.g., at least 2 doses, at least 3 doses, at least 4 doses, at least 5 doses, or more doses) after administration of the donor cells or edited cells. In some embodiments, the agent is administered to the subject in multiple doses (e.g., at least 2 doses, at least 3 doses, at least 4 doses, at least 5 doses, or more doses) before administration of the donor cells or edited cells, and is administered to the subject in multiple doses (e.g., at least 2 doses, at least 3 doses, at least 4 doses, at least 5 doses, or more doses) after administration of the donor cells or edited cells.

[0169] In some embodiments, about 10 6 to 10 11 donor cells or edited cells are administered. In some embodiments, it may be desirable to administer fewer than 10 6 cells to the subject. In some embodiments, it may be desirable to administer more than 10 11 cells to the subject. In some embodiments, one or more doses of cells comprises about 10 6 cells to about 10 11 cells, about 10 7 cells to about 10 10 cells, about 10 8 cells to about 10 9 cells, about 10 6 cells to about 10 8 cells, about 10 7 cells to about 10 9 cells, about 10 7 cells to about 10 10 cells, about 10 7 cells to about 10 11 cells, about 10 8 cells to about 10 10 cells, about 10 8 cells to about 10 11individual cells, approximately 10 9 Individual cells ~ approximately 10 10 individual cells, approximately 10 9 Individual cells ~ approximately 10 11 A single cell, or about 10 10 Individual cells ~ approximately 10 11 Contains 10 cells. In some embodiments, one or more doses of cells are about 10 per kg. 6 ~10 7 Contains individual cells.

[0170] An "antagonist" includes molecules that inhibit the activity of a target protein to any detectable degree. For example, an antagonist of CXCR4 includes molecules that inhibit the activity of CXCR4 (e.g., the binding of CXCR4 to CXCL12 / SDF-1) to any detectable degree.

[0171] In some embodiments, the agent for selective inhibition or depletion of host cells or non-edited cells is an antigen-binding protein.

[0172] The terms "specifically binds" or "binds specifically" mean that an antigen-binding protein (e.g., an antibody or its antigen-binding fragment) binds to an antigen, such as the CXCR4 protein, at least approximately 10 times when measured by a real-time label-free biolayer interferometry assay, for example at 25°C or 37°C, for example by an Octet® HTX biosensor, or by surface plasmon resonance, for example by BIACORE®, or by solution affinity ELISA. -7 M (for example, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 M)'s K DThis refers to having a binding affinity expressed as . In some embodiments, the antigen-binding proteins used herein specifically bind to the CXCR4 protein or human CXCR4 protein (e.g., wild-type or innate CXCR4 protein, e.g., wild-type or innate human CXCR4 protein). "Anti-CXCR4" refers to an antigen-binding protein (or another molecule) that specifically binds to CXCR4, e.g., an antibody or its antigen-binding fragment.

[0173] An antigen is, for example, a molecule to which an antibody binds, such as a peptide (e.g., CXCR4 or a fragment thereof (antigen fragment)). The specific region on the antigen that an antibody recognizes and binds to is called an epitope.

[0174] The term "epitope" refers to a specific antigen-binding site on an antigen-binding protein, for example, an antigenic determinant (e.g., on CXCR4) that interacts with a variable region of an antibody molecule, known as a paratope. A single antigen may have more than one epitopes. Therefore, different antigen-binding proteins (e.g., antibodies) may bind to different regions on an antigen and have different biological effects. The term "epitope" also refers to a site on an antigen to which B cells and / or T cells respond, as well as a region of the antigen to which an antibody binds. Epitopes can be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and have residues that directly contribute to the affinity of the interaction. Epitopes may be linear or conformal, i.e., they may consist of nonlinear amino acids. In certain embodiments, the epitope may include determinants that are chemically active surface groupings of a molecule, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, it may have specific three-dimensional structural features and / or specific charge features. The epitopes to which the antigen-binding proteins used in the present invention bind may be contained in CXCR4, for example, a fragment of human CXCR4, for example, the extracellular loop 2 (ECL2) of human CXCR4 or a part or fragment thereof.

[0175] Methods for determining the epitopes of antigen-binding proteins, such as antibodies, fragments, or polypeptides, include alanine scanning mutation analysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248: 443-63, which is incorporated herein by reference in its entirety), peptide cleavage analysis, crystallographic studies, and NMR analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be utilized (Tomer (2000) Prot. Sci. 9: 487-496, which is incorporated herein by reference in its entirety). Another method that can be used to identify amino acids within polypeptides with which antigen-binding proteins (e.g., antibodies, fragments, or polypeptides) interact is hydrogen / deuterium exchange detected by mass spectrometry. For example, see Ehring (1999) Analytical Biochemistry 267: 252-259 and Engen and Smith (2001) Anal. Chem. 73: 256A-265A, each of which is incorporated herein by reference in its entirety for all purposes.

[0176] The term “antibody,” as used herein, refers to an immunoglobulin molecule (i.e., a “full-length antibody molecule”) (e.g., IgG), e.g., REGN7663 and REGN7664, comprising four polypeptide chains in which two heavy chains (HC) and two light chains (LC) are interconnected by disulfide bonds, e.g., IgG. In some embodiments, the antibody heavy chain comprises SEQ ID NO: 110 or a variant thereof, and the antibody light chain comprises SEQ ID NO: 112 or a variant thereof (REGN7663), or the antibody heavy chain comprises SEQ ID NO: 114 or a variant thereof, and the antibody light chain comprises SEQ ID NO: 116 or a variant thereof (REGN7664). In some embodiments, each antibody heavy chain (HC) comprises a heavy chain variable region ("HCVR" or "V"). H)(For example, for REGN7663, SEQ ID NO: 5 or its variant, and for REGN7664, SEQ ID NO: 21 or its variant) and heavy chain constant region (domain C H 1, C H 2 and C H Each antibody light chain (LC) includes a light chain variable region ("LCVR" or "V"). L )(For example, for REGN7663, SEQ ID NO: 13 or its variant, and for REGN7664, SEQ ID NO: 29 or its variant) and light chain constant region (C L ) includes. V H and V L The region can be further divided into highly variable regions called complementary determination regions (CDRs), within which highly conserved regions called framework regions (FRs) are scattered. H and V L Each comprises three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments, the FRs of the antibody (or its antigen-binding fragment) are identical to human germline sequences or are naturally or artificially modified.

[0177] Typically, the variable domains of both heavy and light immunoglobulin chains contain three hypervariable regions, also called complementarity-determining regions (CDRs), located within a relatively conserved framework region (FR). Generally, in the direction from the N-terminus to the C-terminus, the variable domains of both the light and heavy chains include FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some embodiments, the assignment of amino acids to each domain is as follows: Sequences of Proteins of Immunological Interest, Kabat et al.; National Institutes of Health, Bethesda, Md.; 5 thThe definitions are as follows: ed., NIH Publ. No. 91-3242 (1991), Kabat (1978) Adv. Prot. Chem. 32:1-75, Kabat et al., (1977) J. Biol. Chem. 252:6609-6616, Chothia et al., (1987) J. Mol. Biol. 196:901-917, or Chothia et al., (1989) Nature 342:878-883, each of which is incorporated herein by reference in whole for all purposes. Thus, antigen-binding proteins are, in some embodiments, V H CDR and V L The antibody and antigen-binding fragments include the CDR of V H and V L This includes the amino acid sequences (or variants thereof) described herein, and the CDR is as defined according to Kabat and / or Chothia.

[0178] The terms “antigen-binding portion” or “antigen-binding fragment” of an antibody or antigen-binding protein, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments (the heavy chain portion of a Fab fragment cleaved with papain), and (iv) Fv fragments (V H or V LExamples include (v) single-chain Fv(scFv) molecules (consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR), e.g., CDR3 peptide) or a restricted FR3-CDR3-FR4 peptide). Other manipulated molecules such as domain-specific antibodies, single-domain antibodies, domain deletion antibodies, chimeric antibodies, CDR-transplant antibodies, diabodies, triabodies, tetrabodies, minibodies, and small modular immunotherapy drugs (SMIPs) are also included in the expression “antigen-binding fragment” as used herein. In some embodiments, the antigen-binding fragment comprises three or more CDRs of REGN7663 or REGN7664 (e.g., CDR-H1, CDR-H2, and CDR-H3, or CDR-L1, CDR-L2, and CDR-L3).

[0179] In some embodiments, the antigen-binding protein is a “neutralizing” or “antagonist” anti-target protein antigen-binding protein (e.g., an antibody or antigen-binding fragment) that contains a molecule that inhibits the activity of the target protein to any detectable extent (e.g., inhibits the binding of a receptor to one of its ligands).

[0180] In some embodiments, the antigen-binding protein may include monoclonal antigen-binding proteins, such as antibodies and their antigen-binding fragments, as well as monoclonal compositions comprising multiple isolated monoclonal antigen-binding proteins. The terms “monoclonal antibody” or “mAb,” as used herein, refer to a substantially homogeneous antibody population; that is, the antibody molecules constituting the population have identical amino acid sequences, except for naturally occurring mutations that may be present in trace amounts. The presence of “multiple” such monoclonal antibodies and fragments in a composition refers to an enrichment of antibodies and fragments that are identical (except for naturally occurring mutations that may be present in trace amounts) to those that would naturally be present, for example, in the blood of a host organism, such as a mouse or a human.

[0181] In some embodiments, the antigen-binding protein, e.g., an antibody or antigen-binding fragment, includes, for example, a heavy chain constant domain of the type IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, and IgG4 (including, for example, S228P and / or S108P mutations)), or IgM. In some embodiments, the antigen-binding protein, e.g., an antibody or antigen-binding fragment, includes, for example, a light chain constant domain of the type kappa or lambda. In some embodiments, the antigen-binding protein includes an antigen-binding protein (e.g., REGN7663 or REGN7664) containing a variable domain as described herein, which is linked to the heavy chain and / or light chain constant domain, for example, as described above.

[0182] In some embodiments, the antigen-binding protein is a human antigen-binding protein (e.g., an antibody or its antigen-binding fragment, e.g., REGN7663 or REGN7664). The terms “human” antigen-binding protein, e.g., antibody or antigen-binding fragment, as used herein, include antibodies and fragments having variable and constant regions derived from human germline immunoglobulin sequences, either present in human cells or transplanted into non-human cells, e.g., mouse cells. See, for example, U.S. Patent No. 8502018, No. 6596541, or No. 5789215, each of which is incorporated herein by reference in whole for any purpose. In some embodiments, the human antibody and antigen-binding fragment may include, for example, a CDR, particularly CDR3, with amino acid residues not encoded by a human germline immunoglobulin sequence (e.g., mutations introduced in vitro by random or site-directed mutagenesis, or in vivo by somatic mutation). However, as used herein, the term “human antibody” is not intended to include mAbs in which a CDR sequence derived from the germ cell line of another mammalian species (e.g., mouse) is transplanted onto a human FR sequence. The term includes antibodies produced by recombination in or in non-human mammals. The term is not intended to include antibodies isolated from or produced in human subjects.

[0183] In some embodiments, the antigen-binding protein is a chimeric antigen-binding protein (e.g., a chimeric antibody containing a variable domain as described herein (e.g., derived from REGN7663 or REGN7664)). As used herein, “chimeric antibody” is an antibody having a variable domain derived from a first antibody and a constant domain derived from a second antibody, where the first and second antibodies are derived from different species. See, for example, U.S. Patent No. 4,816,567 and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855, each of which is incorporated herein by reference in whole for all purposes.

[0184] In some embodiments, the antigen-binding protein is a recombinant antigen-binding protein (e.g., a recombinant antigen-binding protein described herein (e.g., REGN7663 or REGN7664)). The term “recombinant” antigen-binding protein, e.g., an antibody or its antigen-binding fragment, refers to a molecule produced, expressed, isolated, or obtained by techniques or methods known in the art as recombinant DNA techniques, including, for example, DNA splicing and transgenic expression. This term includes antibodies expressed in non-human mammals (including transgenic non-human mammals, e.g., transgenic mice), or host cells (e.g., Chinese hamster ovary (CHO) cells), or cell expression systems, or antibodies isolated from recombinant combinatorial human antibody libraries.

[0185] In some embodiments, the antigen-binding protein is an antigen-binding fragment of an antibody (e.g., an antigen-binding fragment of an antigen-binding protein described herein, e.g., REGN7663 or REGN7664). In some embodiments, the antigen-binding fragment of an antibody includes at least one variable domain. The variable domain may be of any size or amino acid composition and generally includes at least one (e.g., three) CDRs adjacent to or in-frame with one or more framework sequences. L V associated with the domain H In an antigen-binding fragment having a domain, V H Domain and V L Domains can be arranged relative to each other in any suitable arrangement. For example, a variable region may be a dimer, V H -V H , V H -V L , or V L -V L It may contain a dimer of . Alternatively, the antigen-binding fragment of the antibody may contain monomer V linked by a non-covalent bond. H Domain and / or V L It may include a domain name.

[0186] In certain embodiments, the antigen-binding fragment of an antibody may include at least one variable domain covalently linked to at least one constant domain. Non-limiting and exemplary configurations of variable and constant domains that may be found within the antigen-binding fragment of an antibody include (i)V H -C H 1. (ii)V H -C H 2. OD V H -C H 3, (iv)V H -C H 1-C H 2. (v)V H -C H 1-C H 2-C H 3. (vi)V H -C H 2-C H 3. (vii)VH -C L , (viii)V L -C H 1, (ix)V L -C H 2, (x)V L -C H 3, (xi)V L -C H 1-C H 2, (xii)V L -C H 1-C H 2-C H 3, (xiii)V L -C H 2-C H 3, and (xiv)V L -C L . In any arrangement of variable domains and constant domains comprising any of the exemplary configurations enumerated above, the variable domains and constant domains may either be directly linked to each other, or may be linked via a complete or partial hinge or linker region. The hinge region may consist of at least 2 amino acids (e.g., 5, 10, 15, 20, 40, 60, or more amino acids), and provides flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, an antigen-binding fragment of an antibody may comprise a homodimer or heterodimer (or other multimer) of any of the above-enumerated configurations of variable and constant domains that are non-covalently associated with each other and / or with one or more monomeric V H or V L domains (e.g., via disulfide bond(s)).

[0187] Antigen-binding proteins (e.g., antibodies and antigen-binding fragments) may be monospecific or multispecific (e.g., bispecific), and may include monospecific antigen-binding fragments and multispecific (e.g., bispecific) antigen-binding fragments comprising one or more variable domains derived from antigen-binding proteins specifically described herein (e.g., REGN7663 or REGN7664).

[0188] In some embodiments, the antigen-binding protein is an antigen-binding protein, such as an antibody (e.g., a human antibody, monoclonal antibody, or recombinant antibody) or its antigen-binding fragment, that specifically binds to the CXCR4 protein or its antigenic fragment (e.g., the extracellular domain of CXCR4 or human CXCR4). For example, the antigen-binding protein may include any polypeptide comprising the amino acid sequence or variant thereof described in SEQ ID NOs. 110, 112, 114, and / or 116. For example, the antigen-binding protein may include any polypeptide comprising the amino acid sequence or variant thereof described in SEQ ID NOs. 5, 13, 21, and / or 29. In another example, the antigen-binding protein may include PF-06747143, urocuplumab, or LY2624587. For example, see Kasyap et al. (2017) J. Hematol. Oncol. 10(1):112 and Peng et al. (2016) PLoS One 11(3):e010585, each of which is incorporated herein by reference in whole for all purposes. Where necessary, the antigen-binding protein includes one or more other polypeptides, e.g., human Fc (e.g., human IgG, e.g., IgG1 or IgG4 (e.g., including the S108P mutation)). Antigen-binding proteins that bind to the same epitope on CXCR4 as any of the antigen-binding proteins described herein (e.g., REGN7663 or REGN7664), or compete for binding to CXCR4, may also be used.

[0189] In some embodiments, the antigen-binding protein (e.g., an antibody or its antigen-binding fragment) comprises a combination of heavy chain CDRs (CDR-H1, CDR-H2, and CDR-H3) as described in SEQ ID NOs. 7, 9, and 11, respectively. H Immunoglobulin heavy chains (e.g., HC) containing and / or combinations of light chain CDRs (CDR-L1, CDR-L2, and CDR-L3) described in SEQ ID NOs. 15, 17, and 19, respectively, are included in V L It contains an immunoglobulin light chain (e.g., LC).

[0190] In some embodiments, the antigen-binding protein (e.g., an antibody or its antigen-binding fragment) comprises, respectively, a combination of heavy and light chain CDRs (CDR-H1, CDR-H2, and CDR-H3, and CDR-L1, CDR-L2, and CDR-L3) as described in SEQ ID NOs. 7, 9, 11, 15, 17, and 19, respectively. H (For example, HC) and V L It includes immunoglobulin heavy and light chains (e.g., LC).

[0191] In some embodiments, the antigen-binding protein (e.g., an antibody or its antigen-binding fragment) comprises a combination of heavy chain CDRs (CDR-H1, CDR-H2, and CDR-H3) as described in SEQ ID NOs. 23, 25, and 27. H Immunoglobulin heavy chains (e.g., HC) containing and / or combinations of light chain CDRs (CDR-L1, CDR-L2, and CDR-L3) described in SEQ ID NOs. 31, 33, and 35, respectively, are included in V L It contains an immunoglobulin light chain (e.g., LC).

[0192] In some embodiments, the antigen-binding protein (e.g., an antibody or its antigen-binding fragment) comprises, respectively, a combination of heavy and light chain CDRs (CDR-H1, CDR-H2, and CDR-H3, and CDR-L1, CDR-L2, and CDR-L3) as described in SEQ ID NOs. 23, 25, 27, 31, 33, and 35, respectively. H(For example, HC) and V L It includes immunoglobulin heavy and light chains (e.g., LC).

[0193] In some embodiments, antigen-binding proteins (e.g., antibodies or their antigen-binding fragments) are, respectively, V H and V L Amino acid sequence: Includes polypeptide pairs, including SEQ ID NOs. 5 and 13.

[0194] In some embodiments, antigen-binding proteins (e.g., antibodies or their antigen-binding fragments) are, respectively, V H and V L Amino acid sequence: Includes a polypeptide pair containing SEQ ID NOs. 21 and 29.

[0195] In some embodiments, the antigen-binding protein (e.g., an antibody or its antigen-binding fragment) is immunoglobulin V H and V L , or including HC and LC, which are the corresponding V as specifically described herein. H , V L The variant amino acid sequences include those having 70% or more (e.g., 80%, 85%, 90%, 95%, 97%, or 99%) overall amino acid sequence identity or similarity to the amino acid sequences of HC, or LC, but CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 of such immunoglobulins are not variants and contain the amino acid sequences described herein. Therefore, in such embodiments, the CDRs within the variant antigen-binding protein are not variants themselves.

[0196] In some embodiments, an antigen-binding protein (e.g., an antibody or its antigen-binding fragment) binds to the same epitope as REGN7663. In some embodiments, an antigen-binding protein (e.g., an antibody or its antigen-binding fragment) competes with REGN7663 for binding to CXCR4. In some embodiments, an antigen-binding protein (e.g., an antibody or its antigen-binding fragment) binds to the same epitope as REGN7664. In some embodiments, an antigen-binding protein (e.g., an antibody or its antigen-binding fragment) competes with REGN7664 for binding to CXCR4. The term "compete," as used herein, means that an antigen-binding protein (e.g., an antibody or its antigen-binding fragment) binds to an antigen (e.g., CXCR4) and inhibits or blocks the binding of another antigen-binding protein (e.g., an antibody or its antigen-binding fragment) to that antigen. Unless otherwise indicated, this term also includes competition between two antigen-binding proteins, e.g., antibodies, in both directions, i.e., a first antibody binding to an antigen and blocking binding by a second antibody, and vice versa. Thus, in some embodiments, competition occurs in one such direction. In certain embodiments, a first antigen-binding protein (e.g., an antibody) and a second antigen-binding protein (e.g., an antibody) may bind to the same epitope. Alternatively, the first and second antigen-binding proteins (e.g., antibodies) may bind to different but overlapping or non-overlapping epitopes, where the binding of one inhibits or blocks the binding of the second antibody, for example, by steric hindrance. Competition between antigen-binding proteins (e.g., antibodies) can be measured by methods known in the art, e.g., by real-time label-free biolayer interferometry assays. Furthermore, binding competition between anti-CXCR4 antigen-binding proteins (e.g., monoclonal antibodies (mAbs)) can be determined using a real-time label-free biolayer interferometry assay with the Octet RED384 biosensor (Pall ForteBio Corp.).

[0197] In some embodiments, the antigen-binding protein is a variant of REGN7663. In some embodiments, the antigen-binding protein is a variant of REGN7664. Typically, a modified antibody or antigen-binding fragment retains the ability to specifically bind to CXCR4, for example, retaining at least 10% of its CXCR4 binding activity (compared to the parent antibody) when its activity is expressed in molar terms. Preferably, the antibody or its antigen-binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100%, or more, of the CXCR4 binding affinity of the parent antibody. It is also intended that the antibody or its antigen-binding fragment may contain conserved or non-conserved amino acid substitutions that do not substantially alter its biological activity (referred to as “conserved variants” or “function-conserved variants” of the antibody).

[0198] Such polypeptides, for example, immunoglobulin chains (e.g., containing the amino acid sequence specifically described herein, of REGN7663) H , V L , or that CDR, or the V of REGN7664 H , V LA "variant" of (or its CDR) is, when compared using the BLAST algorithm, at least about 70-99.9% (e.g., at least 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 8) of the reference amino acid sequence described herein (e.g., any of SEQ ID NOs. 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, or 35) compared to the reference amino acid sequence described herein. This refers to polypeptides containing identical or similar amino acid sequences (6, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9%), where the algorithm parameters are selected to obtain the greatest match between each sequence over the full length of each reference sequence (e.g., prediction threshold: 10, word size: 3, maximum match within query range: 0, BLOSUM 62 matrix, gap cost: present 11, extension 1, conditional compositional score matrix adjustment).

[0199] Furthermore, polypeptide variants may include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) mutations, e.g., one or more missense mutations (e.g., conservative substitutions), nonsense mutations, deletions, or insertions, except for the amino acid sequence of a reference polypeptide specifically described herein, such as an immunoglobulin chain (e.g., HC, LC, V of REGN7663). H , V L , or its CDR, or REGN7664 HC, LC, V H , V L It may include, or its CDR. For example, an immunoglobulin light chain (or V) containing the amino acid sequence described in SEQ ID NO: 13 or 29, but having one or more of such mutations. L) variant, and / or comprises the amino acid sequence set forth in SEQ ID NO: 5 or 21, an immunoglobulin heavy chain having one or more of such mutations (or V H ) An anti-CXCR4 antigen-binding protein comprising a variant can be used in some embodiments. For example, an anti-CXCR4 antigen-binding protein comprising an immunoglobulin light chain variant that comprises the amino acid sequence set forth in SEQ ID NO: 112 or 116 and has one or more of such mutations, and / or an immunoglobulin heavy chain variant that comprises the amino acid sequence set forth in SEQ ID NO: 110 or 114 and has one or more of such mutations, can be used in some embodiments. In some embodiments, the anti-CXCR4 antigen-binding protein comprises an immunoglobulin light chain variant that comprises CDR-L1, CDR-L2, and CDR-L3, wherein one or more (e.g., one, two, or three) of such CDRs has one or more of such mutations (e.g., conservative substitutions), and / or an immunoglobulin heavy chain variant that comprises CDR-H1, CDR-H2, and CDR-H3, wherein one or more (e.g., one, two, or three) of such CDRs has one or more of such mutations (e.g., conservative substitutions).

[0200] The following references relate to the BLAST algorithm, which is often used for sequence analysis: BLAST algorithm: Altschul et al. (2005) FEBS J. 272(20): 5101-5109, Altschul et al. (1990) J. Mol. Biol. 215:403-410, Gish et al. (1993) Nature Genet. 3:266-272, Madden et al. (1996) Meth. Enzymol. 266:131-141, Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402, Zhang et al. (1997) Genome Res. 7:649-656, Wootton et al. (1993) Comput. Chem. 17:149-163, Hancock et al. (1994) Comput. Appl. Biosci. 10:67-70, Alignment scoring system: Dayhoff et al. “A model of evolutionary change in proteins.” in Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3. MO Dayhoff (ed.), pp. 345-352, Natl. Biomed. Res. Found., Washington, DC, Schwartz, RM, et al., “Matrices for detecting distant relationships.” In Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3. MO Dayhoff (ed.), pp. 353-358, Natl. Biomed. Res. Found., Washington, DC, Altschul (1991) J. Mol. Biol. 219:555-565, States et al. (1991) Methods 3:66-70, Henikoff et al. (1992) Proc. Natl.Acad. Sci. USA 89:10915-10919, Altschul et al. (1993) J. Mol. Evol. 36:290-300, Alignment statistics: Karlin et al. (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, Karlin et al. (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877, Dembo et al. (1994) Ann. Prob. 22:2022-2039, and Altschul, “Evaluating the statistical significance of multiple distinct local alignments.” in Theoretical and Computational Methods in Genome Research (S. Suhai, ed.), (1997) pp. 1-14, Plenum, Each of these, including NY, is incorporated herein by reference in its entirety for all purposes.

[0201] For example, a "conservatively modified variant" or "conservative substitution" of an immunoglobulin chain described herein refers to a variant in which one or more amino acids in a polypeptide are substituted with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation, and rigidity, etc.). Such changes can frequently be made without significantly disrupting the biological activity of an antibody or fragment thereof. Those skilled in the art generally recognize that a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.), which is incorporated herein by reference in its entirety for all purposes). In addition, substitutions of structurally or functionally similar amino acids are unlikely to significantly disrupt biological activity. Anti-CXCR4 antigen-binding proteins comprising such conservatively modified variant immunoglobulin chains may be used in some embodiments.

[0202] Examples of groups of amino acids having side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine and methionine. Alternatively, conservative substitutions are any changes that have a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-45, which is incorporated herein by reference in its entirety for all purposes.

[0203] REGN7663, unless otherwise specified, refers to an immunoglobulin heavy chain or its variable region (V) containing the amino acid sequence specifically described herein for REGN7663 (e.g., SEQ ID NO: 5 (or its variant) or SEQ ID NO: 110 (or its variant)). H ) and / or an immunoglobulin light chain or its variable region (V) containing an amino acid sequence specifically described herein for REGN7663 (e.g., SEQ ID NO: 13 (or its variant) or SEQ ID NO: 112 (or its variant)) L ) and / or V containing the heavy chain or its CDR (CDR-H1 (or its variant), CDR-H2 (or its variant), and CDR-H3 (or its variant)) H , and / or V containing the light chain or its CDR (CDR-L1 (or its variant), CDR-L2 (or its variant), and CDR-L3 (or its variant)) L This refers to anti-CXCR4 antigen-binding proteins (e.g., antibodies and their antigen-binding fragments (including multispecific antigen-binding proteins)) that include V. H It is linked to the IgG heavy chain constant domain, for example, the human IgG heavy chain constant domain (e.g., IgG1 or IgG4 (e.g., including S228P and / or S108P mutations)), and / or V L It is linked to a light chain constant domain, for example, a human light chain constant domain (e.g., a lambda or kappa light chain constant domain). In some embodiments, any such immunoglobulin chain (e.g., V H , V L A polynucleotide encoding one or more of the following (, and / or CDR) is provided.

[0204] REGN7664, unless otherwise specified, refers to an immunoglobulin heavy chain or its variable region (V) containing the amino acid sequence specifically described herein for REGN7664 (e.g., SEQ ID NO: 21 (or its variant) or SEQ ID NO: 114 (or its variant)).H ) and / or an immunoglobulin light chain or its variable region (V) containing an amino acid sequence specifically described herein for REGN7664 (e.g., SEQ ID NO: 29 (or its variant) or SEQ ID NO: 116 (or its variant)) L ) and / or V containing the heavy chain or its CDR (CDR-H1 (or its variant), CDR-H2 (or its variant), and CDR-H3 (or its variant)) H , and / or V containing the light chain or its CDR (CDR-L1 (or its variant), CDR-L2 (or its variant), and CDR-L3 (or its variant)) L This refers to anti-CXCR4 antigen-binding proteins (e.g., antibodies and their antigen-binding fragments (including multispecific antigen-binding proteins)) that include V. H It is linked to the IgG heavy chain constant domain, for example, the human IgG heavy chain constant domain (e.g., IgG1 or IgG4 (e.g., including S228P and / or S108P mutations)), and / or V L It is linked to a light chain constant domain, for example, a human light chain constant domain (e.g., a lambda or kappa light chain constant domain). In some embodiments, any such immunoglobulin chain (e.g., V H , V L A polynucleotide encoding one or more of the following (, and / or CDR) is provided.

[0205] In some embodiments, the antigen-binding protein (e.g., an antibody and its antigen-binding fragment (e.g., REGN7663 or REGN7664)) comprises an immunoglobulin chain including the amino acid sequences (and their variants) specifically described herein, as well as post-translational modifications of the antibody or fragment in cells and in vitro. For example, the antigen-binding protein includes an antibody and its antigen-binding fragment that specifically binds to CXCR4 containing the heavy-chain and / or light-chain amino acid sequences described herein, as well as antibodies and fragments having one or more asparagine, serine, and / or threonine residues glycosylated, one or more asparagine residues deamidated, one or more residues (e.g., Met, Trp, and / or His) oxidized, an N-terminal glutamine being pyroglutamate (pyroE), and / or lacking a C-terminal lysine or other amino acid.

[0206] In some embodiments, nucleic acids (may include multiple) encoding antigen-binding proteins are provided. These nucleic acids include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Such nucleic acids may be DNA, RNA, or hybrids or derivatives of either DNA or RNA. Where necessary, in some embodiments, the nucleic acids may be codon-optimized for efficient translation into proteins in specific cells or organisms. In non-limiting examples, nucleic acids may be modified to substitute codons that are more frequently used in human cells, mammalian cells, rodent cells, mouse cells, rat cells, or any other host cells of interest, compared to naturally occurring polynucleotide sequences. Any part or fragment of a nucleic acid molecule may be produced by (1) isolating the molecule from its natural environment, (2) using recombinant DNA technology (e.g., PCR amplification or cloning, but not limited to these), or (3) using chemical synthesis methods. Nucleic acids may be modified to improve stability or reduce immunogenicity. Non-limiting examples of modifications include: (1) alteration or replacement of one or both of the unbound phosphate oxygens and / or one or more of the bound phosphate oxygens in the phosphodiester skeleton linkage; (2) alteration or replacement of components of ribose sugars, e.g., alteration or replacement of the 2' hydroxyl group of ribose sugar; (3) replacement of the phosphate moiety with a dephospholinker; (4) alteration or replacement of naturally occurring nucleic acid bases; (5) replacement or alteration of the ribose-phosphate skeleton; (6) alteration of the 3' or 5' end of an oligonucleotide (e.g., removal, alteration, or replacement of a terminal phosphate group, or partial conjugation); and (7) alteration of sugars.

[0207] Such nucleic acids are operably ligated to promoters or other expression regulatory sequences as needed, e.g., immunoglobulins HC, LC, V of REGN7663 H , V L Immunoglobulins HC, LC, V, which encode CDR-H or CDR-L, or REGN7664H , V L The nucleic acid may include any polynucleotide encoding CDR-H or CDR-L. For example, such nucleic acid may include any polynucleotide (e.g., DNA) containing the nucleotide sequence described in SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 109, 111, 113, or 115. In some embodiments, the polynucleotide of interest is fused to a secretion signal sequence.

[0208] In some embodiments, the nucleic acid may be in the form of an expression construct as defined elsewhere herein. In non-limiting examples, the nucleic acid may include regulatory regions that control the expression of a nucleic acid molecule (e.g., transcriptional or translational regulatory regions), full-length or partial coding regions, and combinations thereof. In non-limiting examples, the nucleic acid may be operably linked to a promoter active in the cell or organism of interest. Promoter options that can be used in such an expression construct include, for example, promoters active in one or more eukaryotic cells, e.g., mammalian cells (e.g., non-human mammalian or human cells), e.g., rodent cells (e.g., mouse or rat cells), etc. Such promoters may be, for example, conditional promoters, inducible promoters, constitutive promoters, or tissue-specific promoters.

[0209] In some embodiments, the nucleic acid(s) include the following polynucleotide pairs encoding HC and LC: SEQ ID NO: 109 and SEQ ID NO: 111. In some embodiments, the nucleic acid(s) include the following polynucleotide pairs encoding HC and LC: SEQ ID NO: 113 and SEQ ID NO: 115. In some embodiments, the nucleic acid(s) include V H and V L Includes the following polynucleotide pairs encoding: SEQ ID NO: 4 and SEQ ID NO: 12. In some embodiments, nucleic acid(s) are V H and V LThe nucleic acid(s) may include the following polynucleotide pairs encoding: SEQ ID NOs. 20 and SEQ ID NOs. 28. In some embodiments, the nucleic acid(s) may include the following polynucleotide sets encoding CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3: SEQ ID NOs. 6, 8, 10, 14, 16, and 18. In some embodiments, the nucleic acid(s) may include the following polynucleotide sets encoding CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3: SEQ ID NOs. 22, 24, 26, 30, 32, and 34. In some embodiments, the nucleic acid(s) may include polynucleotides encoding immunoglobulin polypeptide chains whose nucleotide sequences are variants of those specifically described herein. Such polynucleotide or nucleic acid "variants" refer to polynucleotides or nucleic acids that contain nucleotide sequences that are at least approximately 70–99.9% (e.g., 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9%) identical to the reference nucleotide sequences described herein when compared by the BLAST algorithm, where the algorithm parameters are selected to obtain the greatest match between each sequence over the full length of each reference sequence (e.g., prediction threshold: 10, word size: 28, greatest match in query range: 0, match / mismatch score: 1, -2, gap cost: linear). In some embodiments, variants of nucleotide sequences specifically described herein include one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve) point mutations, insertions (e.g., in-frame insertions), or deletions (e.g., in-frame deletions) of one or more nucleotides. In some embodiments, such mutations may be missense or nonsense mutations.In some embodiments, such variant polynucleotides encode immunoglobulin polypeptide chains that can be incorporated into anti-CXCR4 antigen-binding proteins, i.e., the protein consequently retains specific binding to CXCR4.

[0210] In some embodiments, the antigen-binding protein is an anti-CXCR4 antibody or an antigen-binding fragment thereof. In some embodiments, the antigen-binding protein comprises an immunoglobulin light chain or its variable region containing three light chain CDRs and an immunoglobulin heavy chain or its variable region containing three heavy chain CDRs, wherein each of the three light chain CDRs contains a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 15, 17, and 19, and each of the three heavy chain CDRs contains a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 7, 9, and 11. In some embodiments, the antigen-binding protein comprises an immunoglobulin light chain or its variable region containing three light chain CDRs and an immunoglobulin heavy chain or its variable region containing three heavy chain CDRs, wherein each of the three light chain CDRs consists of a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 15, 17, and 19, and each of the three heavy chain CDRs consists of a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 7, 9, and 11. In some embodiments, the antigen-binding protein comprises an immunoglobulin light chain or its variable region containing three light chain CDRs and an immunoglobulin heavy chain or its variable region containing three heavy chain CDRs, wherein the three light chain CDRs each consist of sequences that are at least 90% identical to the sequences described in SEQ ID NOs. 15, 17, and 19, and the three heavy chain CDRs each consist of sequences that are at least 90% identical to the sequences described in SEQ ID NOs. 7, 9, and 11.

[0211] In some embodiments, the three light chain CDRs each contain the sequences described in SEQ ID NOs. 15, 17, and 19, and the three heavy chain CDRs each contain the sequences described in SEQ ID NOs. 7, 9, and 11. In some embodiments, the three light chain CDRs essentially consist of the sequences described in SEQ ID NOs. 15, 17, and 19, and the three heavy chain CDRs essentially consist of the sequences described in SEQ ID NOs. 7, 9, and 11. In some embodiments, the three light chain CDRs each consist of the sequences described in SEQ ID NOs. 15, 17, and 19, and the three heavy chain CDRs consist of the sequences described in SEQ ID NOs. 7, 9, and 11.

[0212] In some embodiments, the antigen-binding protein is an anti-CXCR4 antibody or an antigen-binding fragment thereof. In some embodiments, the antigen-binding protein comprises an immunoglobulin light chain or its variable region containing three light chain CDRs and an immunoglobulin heavy chain or its variable region containing three heavy chain CDRs, wherein each of the three light chain CDRs contains a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 31, 33, and 35, and each of the three heavy chain CDRs contains a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 23, 25, and 27. In some embodiments, the antigen-binding protein comprises an immunoglobulin light chain or its variable region containing three light chain CDRs and an immunoglobulin heavy chain or its variable region containing three heavy chain CDRs, wherein each of the three light chain CDRs consists of a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 31, 33, and 35, and each of the three heavy chain CDRs consists of a sequence that is at least 90% identical to the sequences described in SEQ ID NOs. 23, 25, and 27. In some embodiments, the antigen-binding protein comprises an immunoglobulin light chain or its variable region containing three light chain CDRs and an immunoglobulin heavy chain or its variable region containing three heavy chain CDRs, wherein the three light chain CDRs each consist of sequences that are at least 90% identical to the sequences described in SEQ ID NOs. 31, 33, and 35, and the three heavy chain CDRs each consist of sequences that are at least 90% identical to the sequences described in SEQ ID NOs. 23, 25, and 27.

[0213] In some embodiments, the three light chain CDRs each contain the sequences described in SEQ ID NOs: 31, 33, and 35, and the three heavy chain CDRs each contain the sequences described in SEQ ID NOs: 23, 25, and 27. In some embodiments, the three light chain CDRs each consist essentially of the sequences described in SEQ ID NOs: 31, 33, and 35, and the three heavy chain CDRs each consist essentially of the sequences described in SEQ ID NOs: 23, 25, and 27. In some embodiments, the three light chain CDRs each consist of the sequences described in SEQ ID NOs: 31, 33, and 35, and the three heavy chain CDRs each consist of the sequences described in SEQ ID NOs: 23, 25, and 27.

[0214] In some embodiments, the immunoglobulin light chain or its variable region includes a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 13, and the immunoglobulin heavy chain or its variable region includes a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 5. In some embodiments, the immunoglobulin light chain or its variable region essentially consists of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 13, and the immunoglobulin heavy chain or its variable region essentially consists of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 5. In some embodiments, the immunoglobulin light chain or its variable region consists of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 13, and the immunoglobulin heavy chain or its variable region consists of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 5.

[0215] In some embodiments, the immunoglobulin light chain or its variable region includes the sequence described in SEQ ID NO: 13, and the immunoglobulin heavy chain or its variable region includes the sequence described in SEQ ID NO: 5. In some embodiments, the immunoglobulin light chain or its variable region is essentially composed of the sequence described in SEQ ID NO: 13, and the immunoglobulin heavy chain or its variable region is essentially composed of the sequence described in SEQ ID NO: 5. In some embodiments, the immunoglobulin light chain or its variable region is composed of the sequence described in SEQ ID NO: 13, and the immunoglobulin heavy chain or its variable region consists of the sequence described in SEQ ID NO: 5.

[0216] In some embodiments, the immunoglobulin light chain comprises a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 112, and the immunoglobulin heavy chain comprises a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 110. In some embodiments, the immunoglobulin light chain consists essentially of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 112, and the immunoglobulin heavy chain consists of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 110. In some embodiments, the immunoglobulin light chain consists of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 112, and the immunoglobulin heavy chain consists of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 110.

[0217] In some embodiments, the immunoglobulin light chain comprises the sequence described in SEQ ID NO: 112, and the immunoglobulin heavy chain comprises the sequence described in SEQ ID NO: 110. In some embodiments, the immunoglobulin light chain is essentially composed of the sequence described in SEQ ID NO: 112, and the immunoglobulin heavy chain is essentially composed of the sequence described in SEQ ID NO: 110. In some embodiments, the immunoglobulin light chain comprises the sequence described in SEQ ID NO: 112, and the immunoglobulin heavy chain comprises the sequence described in SEQ ID NO: 110.

[0218] In some embodiments, the immunoglobulin light chain or its variable region comprises a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 29, and the immunoglobulin heavy chain or its variable region comprises a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 21. In some embodiments, the immunoglobulin light chain or its variable region consists essentially of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 29, and the immunoglobulin heavy chain or its variable region consists of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 21. In some embodiments, the immunoglobulin light chain or its variable region consists of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 29, and the immunoglobulin heavy chain or its variable region consists of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 21.

[0219] In some embodiments, the immunoglobulin light chain or its variable region comprises the sequence described in SEQ ID NO: 29, and the immunoglobulin heavy chain or its variable region comprises the sequence described in SEQ ID NO: 21. In some embodiments, the immunoglobulin light chain or its variable region is essentially composed of the sequence described in SEQ ID NO: 29, and the immunoglobulin heavy chain or its variable region is essentially composed of the sequence described in SEQ ID NO: 21. In some embodiments, the immunoglobulin light chain or its variable region comprises the sequence described in SEQ ID NO: 29, and the immunoglobulin heavy chain or its variable region comprises the sequence described in SEQ ID NO: 21.

[0220] In some embodiments, the immunoglobulin light chain comprises a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 116, and the immunoglobulin heavy chain comprises a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 114. In some embodiments, the immunoglobulin light chain consists of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 116, and the immunoglobulin heavy chain consists of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 114. In some embodiments, the immunoglobulin light chain consists of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 116, and the immunoglobulin heavy chain consists of a sequence that is at least 90% identical to the sequence described in SEQ ID NO: 114.

[0221] In some embodiments, the immunoglobulin light chain comprises the sequence described in SEQ ID NO: 116, and the immunoglobulin heavy chain comprises the sequence described in SEQ ID NO: 114. In some embodiments, the immunoglobulin light chain is essentially composed of the sequence described in SEQ ID NO: 116, and the immunoglobulin heavy chain is essentially composed of the sequence described in SEQ ID NO: 114. In some embodiments, the immunoglobulin light chain comprises the sequence described in SEQ ID NO: 116, and the immunoglobulin heavy chain comprises the sequence described in SEQ ID NO: 114.

[0222] Drugs for selective inhibition or depletion of host cells or non-edited cells may be administered to a subject by any suitable means. The term "administering" refers to the delivery of a composition to a subject or system (e.g., cells, organs, tissues, organisms, or their associated components or sets of components, but not limited to these). The route of administration may vary depending, for example, to the subject or system to which the composition is administered, the nature of the composition, and the purpose of the administration. The terms "administering" or "administering" are intended to include the route through which the drug is introduced to the subject to perform its intended function. Non-limiting examples of routes of administration that may be used in some embodiments include injection (subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal), oral, inhalation, rectal, and transdermal. As a non-limiting example, administration to subjects (e.g., humans or rodents) may be bronchial (including by bronchial drip), buccal, enteral, interdermal, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosa, nose, oral, rectal, subcutaneous, sublingual, topical, trachea (including by intratracheal drip), percutaneous, vaginal, and / or vitreous. The drug may be administered in the form of tablets or capsules (e.g., by injection, inhalation, eye drops, ointment, suppository, etc.), topically by lotion or ointment, or rectally by suppository. Administration may be a bolus or by continuous infusion. Administration may include intermittent or continuous administration (e.g., perfusion) for at least a selected period. Depending on the route of administration, the drug may be coated with or placed within a selected material to protect it from natural conditions that could adversely affect its ability to perform its intended function. The drug may be administered alone or in combination with another drug (e.g., donor cells or edited cells as described herein, but not limited to these) or a pharmaceutically acceptable carrier, or both. The drug may be administered before, simultaneously with, or after the administration of other drugs.Furthermore, the agent may also be administered in a prodrug form that is converted in vivo into its active metabolite or a more active metabolite thereof.

[0223] In some embodiments of the present invention, the subject may include, for example, any type of animal or mammal. Examples of mammals include, but are not limited to, humans, non-human mammals, non-human primates, monkeys, apes, cats, dogs, horses, cattle, deer, bison, sheep, rabbits, rodents (e.g., mice, rats, hamsters, and guinea pigs), and livestock (e.g., but not limited to bovine species such as dairy cows and steers, ovine species such as sheep and goats, and porcine species such as pigs and wild boars). Examples of birds include, but are not limited to, chickens, turkeys, ostriches, geese, and ducks. Domesticated animals and agricultural animals are also included. The term "non-human mammal" excludes humans. Specific non-limiting examples of non-human mammals include rodents, such as mice and rats. In some embodiments of the present invention, the subject is a human.

[0224] In some embodiments of the present invention, any method for improving donor cell engraftment in a subject described herein, or for selective inhibition or depletion of host cells or non-edited cells, may further include the step of generating donor cells or edited cells by modifying a population of cells to express a first isoform of a target protein (e.g., CXCR4 protein). Suitable methods and reagents for generating donor cells or edited cells are described in more detail elsewhere herein. In some embodiments of the present invention, any method for improving donor cell engraftment in a subject described herein, or for selective inhibition or depletion of host cells or non-edited cells, may further include the step of isolating a population of cells from a subject (or from a different subject) before modifying the population of cells to express a first isoform of a target protein (e.g., CXCR4 protein). The isolated cells may be, for example, any suitable cells. In some embodiments, the cells are immune cells. In some embodiments, the cells are hematopoietic cells. In some embodiments, the cells are lymphocytes or lymphoid progenitor cells. In some embodiments, the cells are T cells (e.g., CD4+ T cells, CD8+ T cells, memory T cells, regulatory T cells, gamma delta T cells, mucosa-associated invariant T cells (MAITs), tumor-infiltrating lymphocytes (TILs), or any combination thereof). In some embodiments, the cells are alpha-beta T cells. In some embodiments, the cells are gamma delta T cells. In some embodiments, the cells are TILs. In some embodiments, the cells are B cells. In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are innate lymphoid cells. In some embodiments, the cells are dendritic cells. In some embodiments, the cells are hematopoietic stem cells (HSCs) or hematopoietic stem and progenitor cells (HSPCs) or their offspring.HSCs can give rise to both myeloid and lymphoid progenitor cells, which further give rise to myeloid cells (e.g., monocytes, macrophages, neutrophils, basophils, dendritic cells, erythrocytes, platelets, etc.) and lymphoid cells (e.g., T cells, B cells, NK cells), respectively. In some embodiments, the donor cells or edited cells are autologous (i.e., derived from the subject). In some embodiments, the donor cells or edited cells are allogeneic (i.e., not derived from the subject) or syngeneic (i.e., genetically identical or sufficiently identical, immunologically compatible, and acceptable for transplantation). In some embodiments, the cells are mammalian cells or non-human mammalian cells (e.g., mouse or rat cells or non-human primate cells) (e.g., the subject is a mammal or non-human mammal, and the cells are mammalian cells or non-human mammalian cells). In some embodiments, the cells are human cells (e.g., the subject is a human, and the cells are human cells).

[0225] In some embodiments of the present invention, any method for improving donor cell engraftment in a subject described herein or for selective inhibition or depletion of host cells or non-edited cells may further include the steps of generating donor cells or edited cells by modifying a population of induced pluripotent stem cells (e.g., human induced pluripotent stem cells (iPSCs)) to express a first isoform of a target protein (e.g., CXCR4 protein), and then differentiating the induced pluripotent cells into different cell types before administration to a subject. For example, the induced pluripotent stem cells can be differentiated into any preferred cell type. In some embodiments, the cells are immune cells. In some embodiments, the cells are hematopoietic cells. In some embodiments, the cells are lymphocytes or lymphoid progenitor cells. In some embodiments, the cells are T cells (e.g., CD4+ T cells, CD8+ T cells, memory T cells, regulatory T cells, gamma delta T cells, mucosa-associated invariant T cells (MAITs), tumor-infiltrating lymphocytes (TILs), or any combination thereof). In some embodiments, the cells are alpha-beta T cells. In some embodiments, the cells are gamma-delta T cells. In some embodiments, the cells are TILs. In some embodiments, the cells are B cells. In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are innate lymphoid cells. In some embodiments, the cells are dendritic cells. In some embodiments, the cells are hematopoietic stem cells (HSCs) or hematopoietic stem and progenitor cells (HSPCs) or their offspring. HSCs refer to true stem cells that give rise to all blood and immune lineages. HPSCs include not only HSCs but also more differentiated precursors that give rise to more limited lineages. For example, some HSPCs may only be able to develop into myeloid lineages, or lymphoid lineages, or erythroblasts, etc. HSCs can give rise to both myeloid and lymphoid progenitor cells, which further give rise to myeloid cells (e.g., monocytes, macrophages, neutrophils, basophils, dendritic cells, erythrocytes, platelets, etc.) and lymphoid cells (e.g., T cells, B cells, NK cells), respectively.In some embodiments, the donor cells or edited cells are autologous (i.e., derived from the subject). In some embodiments, the donor cells or edited cells are homogeneous (i.e., not derived from the subject) or syngeneic (i.e., genetically identical or sufficiently identical, immunologically compatible, and acceptable for transplantation). In some embodiments, the cells are mammalian cells or non-human mammalian cells (e.g., mouse or rat cells or non-human primate cells) (e.g., the subject is a mammal or non-human mammal, and the cells are mammalian cells or non-human mammalian cells). In some embodiments, the cells are human cells (e.g., the subject is a human, and the cells are human cells).

[0226] In some embodiments of the present invention, any method for improving donor cell engraftment in a subject as described herein, or for selective inhibition or depletion of host cells or non-edited cells, may further include the steps of generating donor cells or edited cells by modifying a population of hematopoietic stem cells (HSCs) or hematopoietic stem and progenitor cells (HSPCs) (e.g., human HSCs or HSPCs) to express a first isoform of a target protein (e.g., CXCR4 protein), and then differentiating the HSCs or HSPCs into different cell types before administration to a subject. For example, the HSCs or HSPCs can be differentiated into any suitable cell type. In some embodiments, the cells are immune cells. In some embodiments, the cells are hematopoietic cells. In some embodiments, the cells are lymphocytes or lymphoid progenitor cells. In some embodiments, the cells are T cells (e.g., CD4+ T cells, CD8+ T cells, memory T cells, regulatory T cells, gamma delta T cells, mucosa-associated invariant T cells (MAITs), tumor-infiltrating lymphocytes (TILs), or any combination thereof). In some embodiments, the cells are alpha-beta T cells. In some embodiments, the cells are gamma delta T cells. In some embodiments, the cells are TILs. In some embodiments, the cells are B cells. In some embodiments, the cells are immature B cells. In some embodiments, the cells are mature B cells. In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are innate lymphoid cells. In some embodiments, the cells are dendritic cells. In some embodiments, the donor cells or edited cells are autologous (i.e., derived from the subject). In some embodiments, the donor cells or edited cells are homogeneous (i.e., not derived from the subject) or syngeneic (i.e., genetically identical or sufficiently identical, immunologically compatible, and acceptable for transplantation). In some embodiments, the cells are mammalian cells or non-human mammalian cells (e.g., mouse or rat cells or non-human primate cells) (e.g., the subject is a mammal or non-human mammal, and the cells are mammalian cells or non-human mammalian cells).In some embodiments, the cells are human cells (for example, the subject is a human and the cells are human cells). III. Methods for generating donor cells or edited cells

[0227] In some embodiments of the present invention, any method for improving donor cell engraftment in a subject described herein or for selective inhibition or depletion of host cells or unedited cells may further include the step of generating donor cells or edited cells. Donor cells or edited cells can be generated by modifying a cell population to express a first isoform of a target protein (e.g., CXCR4 protein). In some embodiments of the present invention, any method for improving donor cell engraftment in a subject described herein or for selective inhibition or depletion of host cells or unedited cells may further include the step of generating donor cells or edited cells by editing a target genomic locus (e.g., CXCR4 locus) in a cell population to express a first isoform of a target protein (e.g., CXCR4 protein). Donor cells or edited cells may express only the first isoform, or they may express both the first and second isoforms of the target protein (e.g., modified to express the first isoform of the target protein but retaining the expression of the second isoform of the target protein).

[0228] In some embodiments, the step of generating donor or edited cells may include introducing an expression vector into a population of cells, where the expression vector expresses a first isoform of a target protein (e.g., CXCR4 protein). Such an expression vector may comprise the entire coding sequence of the first isoform of the target protein (e.g., CXCR4) operably linked to a promoter suitable for driving expression in the donor or edited cells. Any suitable promoter can be used. In one example, a promoter that is specific to or active in hematopoietic cells or a subset of hematopoietic cells can be used. In another example, a constitutive promoter can be used. Examples of such promoters include human cytomegalovirus (hCMV), chicken beta-actin / CMV enhancer (CAG), and elongation factor-1 alpha (EF1 alpha). In yet another example, an inducible promoter can be used. In some embodiments, the expression vector may be a bicistronic expression vector encoding a therapeutic molecule and a first isoform of a target protein (e.g., CXCR4) and a therapeutic molecule (e.g., CAR, TCR, or an antigen-binding fragment of TCR, or an immunoglobulin), as described elsewhere herein. See, for example, Yeku et al. (2017) Sci. Rep. 7(1):10541 and Rafiq et al. (2018) Nat. Biotechnol. 36(9):847-856, each of which, for any purpose relating to bicistronic constructs expressing, for example, CAR and another molecule, are incorporated herein by reference in their entirety. Any suitable vector may be used. For example, the vector may be a viral vector, such as a lentiviral vector or an adeno-associated virus (AAV) vector. In some embodiments, a lentiviral vector is used. In some embodiments, an AAV vector, such as an AAV vector having a serotype for expression in hematopoietic cells (e.g., AAV6), is used.If necessary, the endogenous locus encoding the second isoform of the target protein may also be modified (e.g., disrupted) so that the first isoform is expressed but the second isoform is not. As one example, the endogenous locus may be modified to include an insertion, deletion, or one or more point mutations in the endogenous locus (e.g., the CXCR4 locus) that results in loss of expression of a functional target protein (e.g., CXCR4). Such a locus may include the deletion or disruption of the entire endogenous coding sequence, or the deletion or disruption of a fragment (i.e., part or portion thereof) of the endogenous locus. As one example, the 5' fragment of the coding sequence may be deleted or disrupted (e.g., including the start codon). As another example, the endogenous locus may be modified so that the start codon of the endogenous locus is deleted, or the start codon is disrupted or mutated in such a way that it is no longer functional. For example, the start codon may be disrupted by a deletion or insertion within the start codon. Alternatively, the start codon may be mutated, for example, by the substitution of one or more nucleotides. In another example, the 3' fragment of the endogenous locus may be deleted or disrupted (e.g., including the stop codon). In yet another example, the internal fragment of the endogenous locus may be deleted or disrupted. In yet another example, the entire coding sequence at the endogenous locus may be deleted or disrupted. Alternatively, the endogenous locus may remain unchanged, and both the first and second isoforms may be expressed.

[0229] In some embodiments, the step of generating donor or edited cells may include editing a genomic locus in a population of cells to express a first isoform of a target protein (e.g., the CXCR4 protein). The genomic locus may be an endogenous locus encoding the target protein, a safe harbor locus, or a random genomic locus targeted by random insertion. A safe harbor locus includes a chromosomal locus in which a transgene or other exogenous nucleic acid insert can be expressed stably and reliably in all tissues of interest without explicitly altering the cell's behavior or phenotype (i.e., without any adverse effects on the host cell). See, for example, Sadelain et al. (2012) Nat. Rev. Cancer 12:51-58, which is incorporated herein by reference in its entirety for all purposes. For example, a safe harbor locus may be one in which the expression of the inserted gene sequence is not disrupted by any read-through expression from adjacent genes. For example, safe harbor loci may include chromosomal loci in which exogenous DNA can be incorporated and function in a predictable manner without adversely affecting the endogenous gene structure or expression. Safe harbor loci may include extragenetic or intragenetic loci, such as those that are not essential, not necessarily required, or can be disrupted without clear phenotypic consequences. Such safe harbor loci can provide open chromatin structure in all tissues and can be ubiquitously expressed during embryonic development and in adulthood. See, for example, Zambrowicz et al. (1997) Proc. Natl. Acad. Sci. USA 94:3789-3794, which is incorporated herein by reference in its entirety for all purposes. In addition, safe harbor loci can be targeted with high efficiency and can be disrupted without clear phenotypes. Examples of safe harbor loci include albumin, CCR5, HPRT, AAVS1, and Rosa26.For example, U.S. Patent Nos. 7,888,121, 7,972,854, 7,914,796, 7,951,925, 8,110,379, 8,409,861, 8,586,526, and U.S. Patent Publications 2003 / 0232410, 2005 / 0208489, 2005 / 0026157, 2006 / 0063231, and 2008 / 0159. See reference to Nos. 996, 2010 / 00218264, 2012 / 0017290, 2011 / 0265198, 2013 / 0137104, 2013 / 0122591, 2013 / 0177983, 2013 / 0177960, and 2013 / 0122591, each of which is incorporated herein by reference in its entirety for all purposes. In some embodiments, the genomic locus is an endogenous genomic locus encoding a target protein (for example, the target protein is CXCR4 and the genomic locus is the CXCR4 genomic locus). In some embodiments, the genomic locus is not an endogenous genomic locus encoding a target protein (for example, the target protein is CXCR4 and the genomic locus is not the CXCR4 genomic locus). The coding sequence of a first isoform of a target protein (e.g., CXCR4) can be operably linked to a promoter suitable for driving expression in donor or edited cells. If necessary, the endogenous locus encoding the second isoform of the target protein can also be modified (e.g., disrupted) so that the first isoform is expressed but the second isoform is not. As an example, the endogenous locus can be modified to include an insertion, deletion, or one or more point mutations in the endogenous locus (e.g., the CXCR4 locus) resulting in loss of expression of the functional target protein (e.g., CXCR4). Such a locus may include the deletion or disruption of the entire endogenous coding sequence, or the deletion or disruption of a fragment (i.e., part or portion thereof) of the endogenous locus. In one example, the 5' fragment of the coding sequence may be deleted or disrupted (e.g., including the start codon).For example, an endogenous locus may be modified such that the start codon of the endogenous locus is deleted or disrupted or mutated in a way that renders it nonfunctional. For example, the start codon may be disrupted by deletion or insertion within the start codon. Alternatively, the start codon may be mutated, for example, by substitution of one or more nucleotides. In another example, the 3' fragment of the endogenous locus may be deleted or disrupted (e.g., including the stop codon). In yet another example, the internal fragment of the endogenous locus may be deleted or disrupted. In yet another example, the entire coding sequence in the endogenous locus may be deleted or disrupted. Alternatively, the endogenous locus may remain unmodified, and both the first and second isoforms may be expressed.

[0230] In some embodiments, the generated donor cells or edited cells may involve editing a target genomic locus (e.g., the CXCR4 locus) to express a first isoform of the target protein (e.g., the CXCR4 protein) in the cell population.

[0231] In some embodiments, the editing step may include introducing into a cell population (1) a nuclease or one or more nucleic acids encoding a nuclease, wherein the nuclease targets a nuclease target sequence at a target genomic locus, and (2) an exogenous donor nucleic acid. The nuclease can cleave the target genomic locus, and the exogenous donor nucleic acid may be inserted into or recombined at the target genomic locus to produce donor cells or edited cells expressing a first isoform of the target protein. However, those skilled in the art will recognize that alternative methods may also be used. For example, in some embodiments, isoform switching may be performed using a base editor. See, for example, Komor et al. (2016) Nature 533(7603):420-424, which is incorporated herein by reference in its entirety for all purposes. This approach allows for the editing of desired amino acids without requiring double-strand DNA breaks.

[0232] Any suitable nuclease agent can be used. In some embodiments, for example, the method may modify a target genomic locus (e.g., the CXCR4 gene, e.g., the human CXCR4 gene) using a nuclease agent, such as a clustered and regularly arranged short palindromic repeat (CRISPR) / CRISPR-related (Cas) system, a zinc finger nuclease (ZFN) system, or a transcription activator-like effector nuclease (TALEN) system, or components of such systems. Generally, the nuclease agent involves the use of a cleavage system engineered to induce double-strand breaks or nicks (i.e., single-strand breaks) at the nuclease target site. Cleavage or nick formation may occur through the use of a specific nuclease, such as an engineered ZFN, TALEN, or CRISPR / Cas system, together with an engineered guide RNA to guide specific cleavage or nick formation at the nuclease target site. Any nuclease agent that induces a nick or double-strand break in a desired target sequence can be used in the methods and compositions disclosed herein. The nuclease agent can be used to generate a targeted gene modification in the CXCR4 gene (e.g., the human CXCR4 gene). For example, in some embodiments, the targeted gene modification may include a targeted gene modification in coding exon 2 of CXCR4 (e.g., coding exon 2 of human CXCR4). In some embodiments, the targeted gene modification is in coding exon 2 of human CXCR4.

[0233] In some embodiments, the nuclease agent is a CRISPR / Cas system. In some embodiments, the nuclease agent comprises one or more ZFNs. In some embodiments, the nuclease agent comprises one or more TALENs.

[0234] The CRISPR / Cas system comprises a transcript and other elements involved in directing the expression or activity of the Cas gene. The CRISPR / Cas system may be, for example, type I, type II, type III, or type V (e.g., subtype VA or subtype VB). The methods and compositions disclosed herein utilize the CRISPR / Cas system by utilizing the CRISPR complex (including a guide RNA (gRNA) complexed with the Cas protein) for site-specific binding or cleavage of nucleic acids. A CRISPR / Cas system targeting a target genomic locus comprises a Cas protein (or a nucleic acid encoding the Cas protein) and one or more guide RNAs (or DNA encoding one or more guide RNAs), each of which targets a different guide RNA target sequence at the target genomic locus.

[0235] The CRISPR / Cas systems used in the compositions and methods disclosed herein may not exist in nature. Non-natural systems include all those that show human involvement, such as those in which one or more components of the system have been altered or mutated from their naturally occurring state, those that do not contain at least substantially one other component that is naturally associated in nature, or those that are associated with at least one other component that is not naturally associated. For example, some CRISPR / Cas systems utilize a non-natural CRISPR complex containing gRNA and Cas protein that do not exist together in nature, or utilize a non-natural Cas protein, or utilize a non-natural gRNA.

[0236] The nuclease agents and CRISPR / Cas systems described in the compositions and methods disclosed herein target a nuclease target sequence (e.g., a guide RNA target sequence) at a target genomic locus encoding a target protein. In some embodiments, the nuclease target sequence is in the CXCR4 gene. In some embodiments, the nuclease target sequence is in the human CXCR4 gene. In some embodiments, the nuclease target sequence is in coding exon 2 of the CXCR4 gene. In some embodiments, the nuclease target sequence is in coding exon 2 of the human CXCR4 gene.

[0237] Cas proteins. Cas proteins generally contain at least one RNA recognition or binding domain that can interact with guide RNA. Cas proteins may also contain nuclease domains (e.g., DNase domains or RNase domains), DNA binding domains, helicase domains, protein-protein interaction domains, dimerization domains, and other domains. Some such domains (e.g., DNase domains) may be derived from innate Cas proteins. Other such domains may be added to create modified Cas proteins. Nuclease domains have catalytic activity for nucleic acid cleavage, which includes the disruption of covalent bonds in nucleic acid molecules. Cleavage can produce blunt or alternating ends, which may be single-stranded or double-stranded. For example, the wild-type Cas9 protein typically yields a blunt cleavage product. Alternatively, the wild-type Cpf1 protein (e.g., FnCpf1) may yield a cleavage product with a 5-nucleotide 5' overhang, where the cleavage occurs after the 18th base pair from the PAM sequence on the untargeted strand and after the 23rd base on the targeted strand. The Cas protein may have complete cleavage activity that generates double-strand breaks at the target genomic locus (e.g., double-strand breaks with blunt ends), or it may be a nickase that generates single-strand breaks at the target genomic locus.

[0238] Examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), C Examples include se4 (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966, as well as their homologs or modified versions.

[0239] Exemplary Cas proteins are Cas9 proteins or proteins derived from Cas9 proteins. Cas9 proteins originate from the type II CRISPR / Cas system and typically share four key motifs with a conserved architecture. Motifs 1, 2, and 4 are RuvC-like motifs, and motif 3 is an HNH motif. Exemplary Cas9 proteins are Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira Derived from sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Acaryochloris marina, Neisseria meningitidis, or Campylobacter jejuni. Examples of additional Cas9 family members are described in WO2014 / 131833, which is incorporated herein by reference in its entirety for all purposes. S.Cas9 derived from pyogenes (SpCas9) (e.g., assigned UniProt accession number Q99ZW2) is an exemplary Cas9 protein. The exemplary SpCas9 protein sequence is described in SEQ ID NO: 36 (encoded by the DNA sequence described in SEQ ID NO: 37). Smaller Cas9 proteins (e.g., Cas9 proteins whose coding sequence, when combined with a guide RNA coding sequence and regulatory elements for Cas9 and guide RNA, is compatible with the maximum AAV packaging capacity, e.g., SaCas9, CjCas9, and Nme2Cas9) are other exemplary Cas9 proteins. For example, Cas9 derived from S. aureus (SaCas9) (e.g., assigned UniProt accession number J7RUA5) is another exemplary Cas9 protein. Similarly, Cas9 derived from Campylobacter jejuni (CjCas9) (e.g., assigned UniProt accession number Q0P897) is another exemplary Cas9 protein. For example, see Kim et al. (2017) Nat. Commun. 8:14500, which is incorporated herein by reference in its entirety for all purposes. SaCas9 is smaller than SpCas9, and CjCas9 is smaller than both SaCas9 and SpCas9. Cas9 (Nme2Cas9) derived from Neisseria meningitidis is another exemplary Cas9 protein. For example, see Edraki et al. (2019) Mol.See Cell 73(4):714–726, which is incorporated herein by reference in its entirety for all purposes. Cas9 proteins derived from Streptococcus thermophilus (e.g., Streptococcus thermophilus LMD-9 Cas9 (St1Cas9) encoded by the CRISPR1 locus or Streptococcus thermophilus Cas9 (St3Cas9) derived from the CRISPR3 locus) are other exemplary Cas9 proteins. Cas9 derived from Francisella novicida (FnCas9) or the RHA Francisella novicida Cas9 variant, which recognizes alternative PAM (E1369R / E1449H / R1556A substitution), are other exemplary Cas9 proteins. These and other exemplary Cas9 proteins are outlined, for example, in Cebrian-Serrano and Davies (2017) Mamm. Genome 28(7):247-261, which is incorporated herein by reference in its entirety for all purposes. Examples of Cas9 coding sequences, Cas9 mRNA, and Cas9 protein sequences are provided in WO2013 / 176772, WO2014 / 065596, WO2016 / 106121, WO2019 / 067910, WO2020 / 082042, US2020 / 0270617, WO2020 / 082041, US2020 / 0268906, WO2020 / 082046, and US2020 / 0289628, each of which is incorporated herein by reference in its entirety for all purposes. Specific examples of ORF and Cas9 amino acid sequences are provided in Table 30 of paragraph

[0449] of WO2019 / 067910, and specific examples of Cas9 mRNA and ORF are provided in paragraphs

[0214] to

[0234] of WO2019 / 067910. See also Table 24 of WO2020 / 082046A2 (pp. 84-85) and WO2020 / 069296, each of which is incorporated herein by reference in its entirety for all purposes.

[0240] Another example of a Cas protein is the Cpf1 (CRISPR, Cas12a, derived from Prevotella and Francisella 1) protein. Cpf1 is a large protein (approximately 1300 amino acids) containing a RuvC-like nuclease domain homologous to the corresponding domain of Cas9, along with a counterpart to the characteristic arginine-rich cluster of Cas9. However, in contrast to Cas9, which contains a long insert containing an HNH domain, Cpf1 lacks the HNH nuclease domain present in the Cas9 protein, and the RuvC-like domain is adjacent in the Cpf1 sequence. See, for example, Zetsche et al. (2015) Cell 163(3):759-771, which is incorporated herein by reference in its entirety for all purposes. Exemplary Cpf1 proteins are Francisella tularensis 1, Francisella tularensis subsp. GW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3. Derived from Prevotella disiens and Porphyromonas macacae. Cpf1 (FnCpf1, assigned UniProt accession number A0Q7Q2) derived from Francisella novicida U112 is an exemplary Cpf1 protein.

[0241] Another example of a Cas protein is CasX (Cas12e). CasX is an RNA-guided DNA endonuclease that produces alternating double-strand breaks in DNA. CasX is smaller than 1000 amino acids. Exemplary CasX proteins are derived from Deltaproteobacteria (DpbCasX or DpbCas12e) and Plantomycetes (PlmCasX or PlmCas12e). Similar to Cpf1, CasX uses a single RuvC active site for DNA cleavage. See, for example, Liu et al. (2019) Nature 566(7743):218-223, which is incorporated herein by reference in its entirety for all purposes.

[0242] Another example of a Cas protein is CasΦ (CasPhi or Cas12j), which is uniquely found in bacteriophages. CasΦ is the size of fewer than 1000 amino acids (e.g., 700–800 amino acids). CasΦ cleavage produces alternating 5' overhangs. A single RuvC active site in CasΦ is capable of crRNA processing and DNA cleavage. See, for example, Pausch et al. (2020) Science 369(6501):333–337, which is incorporated herein by reference in its entirety for all purposes.

[0243] The Cas protein may be a wild-type protein (i.e., one that occurs in nature), a modified Cas protein (i.e., a Cas protein variant), or a fragment of a wild-type or modified Cas protein. The Cas protein may also be a variant or fragment that is active with respect to the catalytic activity of a wild-type or modified Cas protein. A variant or fragment that is active with respect to catalytic activity may contain at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with respect to a wild-type or modified Cas protein or a portion thereof, and the active variant retains the ability to cleave at a desired cleavage site and therefore retains nick-inducing or double-strand break-inducing activity. Assays for nick-inducing or double-strand break-inducing activity are known and generally measure the overall activity and specificity of the Cas protein in a DNA substrate containing a cleavage site.

[0244] One example of a modified Cas protein is the modified SpCas9-HF1 protein, which is a high-fidelity variant of Streptococcus pyogenes Cas9 with modifications (N497A / R661A / Q695A / Q926A) designed to reduce nonspecific DNA contact. See, for example, Kleinstiver et al. (2016) Nature 529(7587):490-495, which is incorporated herein by reference in its entirety for all purposes. Another example of a modified Cas protein is the modified eSpCas9 variant (K848A / K1003A / R1060A) designed to reduce off-target effects. See, for example, Slaymaker et al. (2016) Science 351(6268):84-88, which is incorporated herein by reference in its entirety for all purposes. Other SpCas9 variants include K855A and K810A / K1003A / R1060A. These and other modified Cas proteins are outlined, for example, in Cebrian-Serrano and Davies (2017) Mamm. Genome 28(7):247-261, which is incorporated herein by reference in its entirety for all purposes. Another example of a modified Cas9 protein is xCas9, which is a SpCas9 variant capable of recognizing an expanded PAM sequence range. See, for example, Hu et al. (2018) Nature 556:57-63, which is incorporated herein by reference in its entirety for all purposes.

[0245] Cas proteins can be modified to increase or decrease one or more of the following: nucleic acid binding affinity, nucleic acid binding specificity, and enzymatic activity. Cas proteins can also be modified to alter any other activity or property of the protein, such as stability. For example, one or more nuclease domains of a Cas protein may be modified, deleted, or inactivated, or a Cas protein may be shortened to remove domains that are not essential to the protein's function, or to optimize (e.g., enhance or reduce) the activity or property of the Cas protein.

[0246] Cas proteins may contain at least one nuclease domain, e.g., a DNase domain. For example, the wild-type Cpf1 protein generally contains a RuvC-like domain that cleaves both strands of target DNA, possibly in a dimeric configuration. Similarly, CasX and CasΦ generally contain a single RuvC-like domain that cleaves both strands of target DNA. Cas proteins may also contain at least two nuclease domains, e.g., DNase domains. For example, the wild-type Cas9 protein generally contains a RuvC-like nuclease domain and an HNH-like nuclease domain. The RuvC and HNH domains can each cleave different strands of double-stranded DNA to create double-strand breaks in the DNA. See, for example, Jinek et al. (2012) Science 337(6096):816-821, which is incorporated herein by reference in its entirety for all purposes.

[0247] One or more nuclease domains may be deleted or mutated so that they are no longer functional or have reduced nuclease activity. For example, if one of the nuclease domains is deleted or mutated in the Cas9 protein, the resulting Cas9 protein can be called a nickase, which can produce single-strand breaks in double-stranded target DNA but not double-strand breaks (i.e., it can cleave the complementary or non-complementary strand, but not both). If all nuclease domains are neither deleted nor mutated in the Cas9 protein, the Cas9 protein retains double-strand break-inducing activity. An example of a mutation that converts Cas9 to a nickase is the D10A mutation in the RuvC domain of Cas9 from S. pyogenes (where the aspartic acid at position 10 of Cas9 becomes alanine). Similarly, Cas9 can be converted to nickase by H939A (hitidine at amino acid position 839 becomes alanine), H840A (hitidine at amino acid position 840 becomes alanine), or N863A (asparagine at amino acid position N863 becomes alanine) in the HNH domain of Cas9 derived from S. pyogenes. Other examples of Cas9-nickase-converting mutations include corresponding mutations for Cas9 derived from S. thermophilus. See, for example, Sapranauskas et al. (2011) Nucleic Acids Res. 39(21):9275-9282 and WO2013 / 141680, each of which is incorporated herein by reference in whole for all purposes. Such mutations can be generated using methods such as site-directed mutagenesis, PCR-mediated mutagenesis, or whole-gene synthesis. Other examples of mutations that produce nickase can be found, for example, in WO2013 / 176772 and WO2013 / 142578, each of which is incorporated herein by reference in its entirety for all purposes.

[0248] Examples of inactivating mutations in the catalytic domain of xCas9 are the same as those for SpCas9 described above. Examples of inactivating mutations in the catalytic domain of the Staphylococcus aureus Cas9 protein are also known. For example, the Staphylococcus aureus Cas9 enzyme (SaCas9) may contain substitutions at position N580 (e.g., N580A substitution) or at position D10 (e.g., D10A substitution) to produce Cas nickase. See, for example, WO2016 / 106236, which is incorporated herein by reference in its entirety for all purposes. Examples of inactivating mutations in the catalytic domain of Nme2Cas9 are also known (e.g., D16A or H588A). Examples of inactivating mutations in the catalytic domain of St1Cas9 are also known (e.g., D9A, D598A, H599A, or N622A). Examples of inactivating mutations in the catalytic domain of St3Cas9 are also known (e.g., D10A or N870A). Examples of inactivating mutations in the catalytic domain of CjCas9 are also known (e.g., the combination of D8A or H559A). Examples of inactivating mutations in the catalytic domain of FnCas9 and RHA are also known (e.g., N995A).

[0249] Examples of inactivating mutations in the catalytic domain of the Cpf1 protein are also known. Referring to the Cpf1 proteins derived from Francisella novicida U112 (FnCpf1), Acidaminococcus sp. BV3L6 (AsCpf1), Lachnospiraceae bacterium ND2006 (LbCpf1), and Moraxella bovoculi 237 (MbCpf1 Cpf1), such mutations may include mutations at positions 908, 993, or 1263 in AsCpf1, or the corresponding positions in the Cpf1 ortholog, or at positions 832, 925, 947, or 1180 in LbCpf1, or the corresponding positions in the Cpf1 ortholog. Such mutations may include, for example, one or more of the mutations D908A, E993A, and D1263A in AsCpf1, or the corresponding mutations in Cpf1 orthologues, or D832A, E925A, D947A, and D1180A in LbCpf1, or the corresponding mutations in Cpf1 orthologues. See, for example, US2016 / 0208243, which is incorporated herein by reference in its entirety for all purposes. Examples of inactivating mutations in the catalytic domain of the CasX protein are also known. With reference to the CasX protein derived from Deltaproteobacteria, D672A, E769A, and D935A (individually or in combination), or the corresponding positions in other CasX orthologues, are inactivating. For example, see Liu et al. (2019) Nature 566(7743):218-223, which is incorporated herein by reference in its entirety for all purposes. Examples of inactivating mutations in the catalytic domain of the CasΦ protein are also known. For example, D371A and D394A are inactivating mutations, either individually or in combination. For example, see Pausch et al. (2020) Science 369(6501):333-337, which is incorporated herein by reference in its entirety for all purposes.

[0250] The Cas protein can also be operably linked to a heterologous polypeptide as a fusion protein. For example, the Cas protein can be fused to a cleavage domain. See WO2014 / 089290, which is incorporated herein by reference in its entirety for all purposes. The Cas protein can also be fused to a heterologous polypeptide to provide increased or decreased stability. The fusion domain or heterologous polypeptide may be located at the N-terminus, C-terminus, or internally within the Cas protein.

[0251] As one example, a Cas protein may be fused with one or more heterologous polypeptides that provide intracellular localization. Such heterologous polypeptides may include, for example, one or more nuclear localization signals (NLS) for directing the signal to the nucleus, e.g., monopartite SV40 NLS and / or bipartite alpha-importin NLS, mitochondrial localization signals for directing the signal to mitochondria, ER retention signals, etc. See, for example, Lange et al. (2007) J. Biol. Chem. 282(8):5101-5105, which is incorporated herein by reference in its entirety for all purposes. Such intracellular localization signals may be located at the N-terminus, C-terminus, or anywhere else within the Cas protein. The NLS may consist of a sequence of basic amino acids and may be a monopartite or bipartite sequence. If necessary, Cas proteins may contain two or more NLSs, including an NLS at the N-terminus (e.g., an alpha-importin NLS or a monosegmental NLS) and an NLS at the C-terminus (e.g., an SV40 NLS or a bisegmental NLS). Cas proteins may also contain two or more NLSs at the N-terminus and / or two or more NLSs at the C-terminus.

[0252] The Cas protein can be fused with, for example, 1 to 10 NLSs (e.g., 1 to 5 NLSs, or 1 NLS). If one NLS is used, the NLS can be ligated to the N-terminus or C-terminus of the Cas protein sequence. This can also be inserted into the Cas protein sequence. Alternatively, the Cas protein can be fused with more than one NLS. For example, the Cas protein can be fused with 2, 3, 4, or 5 NLSs. In a specific example, the Cas protein can be fused with 2 NLSs. In certain circumstances, the two NLSs may be the same (e.g., 2 SV40 NLSs) or different. For example, the Cas protein may be fused with 2 SV40 NLSs ligated to the carboxyl terminus. The Cas protein can be fused to an NLS sequence. Alternatively, the Cas protein can be fused to two NLS sequences, one linked to the N-terminus and the other to the C-terminus. In other examples, the Cas protein may be fused to three NLS sequences, or not fused to any NLS sequences at all. The NLS can be a monosegmental sequence, e.g., SV40 NLS, PKKKRKV (SEQ ID NO: 38), or PKKKRRV (SEQ ID NO: 39). The NLS can be a bisegmental sequence, e.g., the nucleoplasmin NLS, KRPAATKKAGQAKKKK (SEQ ID NO: 40). In a specific example, a single PKKKRKV (SEQ ID NO: 38) NLS can be linked to the C-terminus of the Cas protein. One or more linkers may be included in the fusion site, as required.

[0253] The Cas protein can also be operably linked to a cell permeability domain or a protein transduction domain. For example, the cell permeability domain may be derived from the HIV-1 TAT protein, the TLM cell permeability motif derived from human hepatitis B virus, MPG, Pep-1, VP22, a cell permeability peptide derived from herpes simplex virus, or a polyarginine peptide sequence. See, for example, WO2014 / 089290 and WO2013 / 176772, each of which is incorporated herein by reference in whole for any purpose. The cell permeability domain may be located at the N-terminus, C-terminus, or anywhere else within the Cas protein.

[0254] The Cas protein can be provided in any form. For example, the Cas protein can be provided in the form of a Cas protein complexed with a protein, such as gRNA. Alternatively, the Cas protein can be provided in the form of a nucleic acid encoding the Cas protein, such as RNA (e.g., messenger RNA (mRNA)) or DNA. If necessary, the nucleic acid encoding the Cas protein can be codon-optimized for efficient translation into the protein in a particular cell or organism. For example, the nucleic acid encoding the Cas protein can be modified to substitute codons that are frequently used in bacterial cells, yeast cells, human cells, non-human cells, mammalian cells, rodent cells, mouse cells, rat cells, or any other host cell of interest, compared to a naturally occurring polynucleotide sequence. Once the nucleic acid encoding the Cas protein is introduced into a cell, the Cas protein can be expressed transiently, conditionally, or constitutively in the cell.

[0255] The nucleic acid encoding the Cas protein can be stably integrated into the cell's genome and operably linked to an active promoter in the cell. Alternatively, the nucleic acid encoding the Cas protein can be operably linked to a promoter in an expression construct. An expression construct comprises any nucleic acid construct that can direct the expression of a target gene or other nucleic acid sequence (e.g., the Cas gene) and transfer such a target nucleic acid sequence into a target cell. For example, the nucleic acid encoding the Cas protein may be in a vector containing DNA encoding a gRNA. Alternatively, it may be in a vector or plasmid separate from the vector containing DNA encoding the gRNA. Promoters that can be used in an expression construct include, for example, promoters active in human cells or human hematopoietic cells. Such promoters may be, for example, conditional promoters, inductive promoters, constitutive promoters, or tissue-specific promoters. If necessary, the promoter may be a bidirectional promoter that drives the expression of both the Cas protein in one direction and the guide RNA in the other direction. Such bidirectional promoters may consist of (1) a complete conventional unidirectional Pol III promoter containing three external regulatory elements: a distal sequence element (DSE), a proximal sequence element (PSE), and a TATA box, and (2) a second basic Pol III promoter containing a PSE and a TATA box fused to the 5' end of the DSE in the reverse orientation. For example, in the H1 promoter, the promoter can be made bidirectional by creating a hybrid promoter in which the DSE is adjacent to the PSE and TATA box, and reverse transcription is controlled by the addition of a PSE and TATA box derived from the U6 promoter. See, for example, US2016 / 0074535, which is incorporated herein by reference in its entirety for all purposes. By using bidirectional promoters, it is possible to create a small expression cassette that facilitates delivery by simultaneously expressing the gene encoding the Cas protein and guide RNA.In certain embodiments, the promoter is accepted by regulatory authorities for use in humans. In certain embodiments, the promoter drives expression in hematopoietic cells.

[0256] Cas expression or Cas9 expression can be driven using different promoters. In some methods, small promoters are used so that the Cas or Cas9 coding sequence can be fitted to an AAV construct. For example, Cas or Cas9 and one or more gRNAs (e.g., one, two, three, or four gRNAs) can be delivered via LNP-mediated delivery (e.g., in RNA form) or adeno-associated virus (AAV)-mediated delivery (e.g., AAV8-mediated delivery). For example, a nuclease agent may be CRISPR / Cas9, and Cas9 mRNA and gRNA (e.g., targeting the CXCR4 gene (e.g., the human CXCR4 gene)) can be delivered via LNP-mediated delivery or AAV-mediated delivery. Cas or Cas9 and gRNA(s) can be delivered by a single AAV or via two separate AAVs. For example, a first AAV may carry a Cas or Cas9 expression cassette, and a second AAV may carry a gRNA expression cassette. Similarly, a first AAV may carry a Cas or Cas9 expression cassette, and a second AAV may carry two or more gRNA expression cassettes. Alternatively, a single AAV may carry a Cas or Cas9 expression cassette (e.g., a Cas or Cas9 coding sequence operably ligated to a promoter) and a gRNA expression cassette (e.g., a gRNA coding sequence operably ligated to a promoter). Similarly, a single AAV may carry a Cas or Cas9 expression cassette (e.g., a Cas or Cas9 coding sequence operably ligated to a promoter) and two or more gRNA expression cassettes (e.g., gRNA coding sequences operably ligated to a promoter). Different promoters, e.g., the U6 promoter or a small tRNA Gln, may be used to drive gRNA expression. Similarly, different promoters may be used to drive Cas9 expression. For example, a small promoter is used so that the Cas9 coding sequence can be fitted into the AAV construct.Similarly, small Cas9 proteins (e.g., SaCas9 or CjCas9) are used to maximize AAV packaging capacity.

[0257] Cas proteins provided as mRNA may be modified to improve their stability and / or immunogenicity. Modifications may be made to one or more nucleosides within the mRNA. Additionally, the mRNA encoding the Cas protein may be capped. The Cas mRNA may further contain a polyadenylated (poly-A or poly(A) or poly-adenine) tail. For example, the Cas mRNA may contain modifications to one or more nucleosides within the mRNA, the Cas mRNA may be capped, and the Cas mRNA may contain a poly(A) tail.

[0258] Guide RNA. A "guide RNA" or "gRNA" is an RNA molecule that binds to a Cas protein (e.g., the Cas9 protein) and directs the Cas protein to a specific location within target DNA. A guide RNA may contain two segments: a "DNA targeting segment" (also called a "guide sequence") and a "protein-binding segment." A "segment" is a division or region of a molecule, for example, a continuous sequence of nucleotides in RNA. Some gRNAs, for example, those for Cas9, may contain two distinct RNA molecules: an "activator-RNA" (e.g., tracrRNA) and a "targeter-RNA" (e.g., CRISPR RNA or crRNA). Other gRNAs are single RNA molecules (single RNA polynucleotides), which can also be called "single-molecule gRNA," "single-guide RNA," or "sgRNA." For example, see WO2013 / 176772, WO2014 / 065596, WO2014 / 089290, WO2014 / 093622, WO2014 / 099750, WO2013 / 142578, and WO2014 / 131833, each of which is incorporated herein by reference in its entirety for any purpose. Guide RNA may refer to either CRISPR RNA (crRNA) or a combination of crRNA and transactivated CRISPR RNA (tracrRNA). crRNA and tracrRNA may associate as a single RNA molecule (single guide RNA or sgRNA) or as two separate RNA molecules (dual guide RNA or dgRNA). For Cas9, for example, single guide RNA may include crRNA fused to tracrRNA (e.g., via a linker). For Cpf1 and CasΦ, for example, only crRNA is required to achieve binding to the target sequence. The terms “guide RNA” and “gRNA” include both bimolecule (i.e., modular) gRNA and monomolecule gRNA. In some of the methods and compositions disclosed herein, the gRNA is S. pyogenes Cas9 gRNA or its equivalent.In some of the methods and compositions disclosed herein, the gRNA is S. aureus Cas9 gRNA or its equivalent.

[0259] The two exemplary gRNA molecules include a crRNA-like molecule ("CRISPR RNA," "targeter-RNA," "crRNA," or "crRNA repeat") and a corresponding tracrRNA-like molecule ("transactivating CRISPR RNA," "activator-RNA," or "tracrRNA"). The crRNA contains both the DNA targeting segment (single-stranded) of the gRNA and a sequence of nucleotides forming one of the dsRNA double helix of the protein-binding segment of the gRNA. An example of a crRNA tail located downstream (3') of the DNA targeting segment (e.g., for use in S. pyogenes Cas9) is: [ka] or [ka] It includes, is essentially derived from, or consists of. Any of the DNA targeting segments disclosed herein can bind to the 5' end of SEQ ID NO: 41 or 42 to form a crRNA.

[0260] The corresponding tracrRNA (activator-RNA) contains a sequence of nucleotides that form the other half of the dsRNA double helix of the protein-binding segment of the gRNA. The sequence of nucleotides in the crRNA is complementary to the sequence of nucleotides in the tracrRNA, and they hybridize to form the dsRNA double helix of the protein-binding domain of the gRNA. Therefore, it can be said that each crRNA has a corresponding tracrRNA. An example of a tracrRNA sequence (e.g., for use in S. pyogenes Cas9) is: [ka] It includes, essentially consists of, or comprises one of the following.

[0261] In systems requiring both crRNA and tracrRNA, the crRNA and its corresponding tracrRNA hybridize to form a gRNA. In systems requiring only crRNA, the crRNA may be a gRNA. The crRNA further provides a single-stranded DNA targeting segment that hybridizes to the complementary strand of the target DNA. When used for intracellular modification, the precise sequence of a given crRNA or tracrRNA molecule can be designed to be species-specific in which the RNA molecule is used. For example, see Mali et al. (2013) Science 339(6121):823-826, Jinek et al. (2012) Science 337(6096):816-821, Hwang et al. (2013) Nat. Biotechnol. 31(3):227-229, Jiang et al. (2013) Nat. Biotechnol. 31(3):233-239, and Cong et al. (2013) Science 339(6121):819-823, each of which is incorporated herein by reference in its entirety for all purposes.

[0262] The DNA targeting segment (crRNA) of a given gRNA contains a nucleotide sequence complementary to the sequence of the complementary strand of the target DNA, as described in more detail below. The DNA targeting segment of the gRNA interacts with the target DNA in a sequence-specific manner via hybridization (i.e., base pairing). Therefore, the nucleotide sequence of the DNA targeting segment can vary, determining the position within the target DNA where the gRNA and target DNA interact. The DNA targeting segment of a target gRNA can be modified to hybridize to any desired sequence within the target DNA. Naturally occurring crRNAs, depending on the CRISPR / Cas system and the organism, often contain a targeting segment of 21 to 72 nucleotides in length, flanked by two direct repeats (DRs) of 21 to 46 nucleotides in length (see, for example, WO2014 / 131833, which is incorporated herein by reference in its entirety for all purposes). In the case of S. pyogenes, the DR is 36 nucleotides long, and the targeting segment is 30 nucleotides long. The DR, positioned at 3', is complementary to the corresponding tracrRNA and hybridizes with it, which then binds to the Cas protein.

[0263] A DNA targeting segment may have a length of, for example, at least about 12, at least about 15, at least about 17, at least about 18, at least about 19, at least about 20, at least about 25, at least about 30, at least about 35, or at least about 40 nucleotides. Such a DNA targeting segment may have a length of, for example, about 12 to about 100, about 12 to about 80, about 12 to about 50, about 12 to about 40, about 12 to about 30, about 12 to about 25, or about 12 to about 20 nucleotides. For example, a DNA targeting segment may have about 15 to about 25 nucleotides (e.g., about 17 to about 20 nucleotides, or about 17, 18, 19, or 20 nucleotides). See, for example, US2016 / 0024523, which is incorporated herein by reference in its entirety for all purposes. For Cas9 derived from S. pyogenes, the typical DNA targeting segment is 16–20 nucleotides long, or 17–20 nucleotides long. For Cas9 derived from S. aureus, the typical DNA targeting segment is 21–23 nucleotides long. For Cpf1, the typical DNA targeting segment is at least 16 nucleotides long, or at least 18 nucleotides long.

[0264] In one example, the DNA targeting segment may be approximately 20 nucleotides long. However, shorter and longer sequences can also be used as targeting segments (e.g., 15–25 nucleotides long, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides long). The degree of identity between the DNA targeting segment and the corresponding guide RNA target sequence (or the degree of complementarity between the DNA targeting segment and the other strand of the guide RNA target sequence) may be, for example, approximately 75%, 80%, 85%, 90%, 95%, or 100%. The DNA targeting segment and the corresponding guide RNA target sequence may contain one or more mismatches. For example, the DNA targeting segment of the guide RNA and the corresponding guide RNA target sequence may contain 1 to 4, 1 to 3, 1 to 2, 1, 2, 3, or 4 mismatches (for example, if the total length of the guide RNA target sequence is at least 17, at least 18, at least 19, or at least 20, or more nucleotides). For example, if the total length of the guide RNA target sequence is 20 nucleotides, the DNA targeting segment of the guide RNA and the corresponding guide RNA target sequence may contain 1 to 4, 1 to 3, 1 to 2, 1, 2, 3, or 4 mismatches.

[0265] As one example, a guide RNA targeting human CXCR4 (e.g., human CXCR4 coding exon 2) may include a DNA targeting segment (i.e., a guide sequence) that includes, is essentially derived from, or consists of the sequence (DNA targeting segment) described in any one of sequence numbers 132-146. Alternatively, a guide RNA targeting human CXCR4 (e.g., human CXCR4 coding exon 2) may include a DNA targeting segment that includes, is essentially derived from, or consists of at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides of the sequence (DNA targeting segment) described in any one of sequence numbers 132-146. Alternatively, a guide RNA targeting human CXCR4 (e.g., human CXCR4 coding exon 2) may include a DNA targeting segment that is at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the sequence (DNA targeting segment) described in any one of sequence numbers 132-146. Alternatively, a guide RNA targeting human CXCR4 (e.g., human CXCR4 coding exon 2) may include a DNA targeting segment that is at least 90% or at least 95% identical to the sequence (DNA targeting segment) described in any one of sequence numbers 132-146. Alternatively, a guide RNA targeting human CXCR4 (e.g., human CXCR4 coding exon 2) may include a DNA targeting segment that is at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides of the sequence (DNA targeting segment) described in any one of sequence numbers 132-146.Alternatively, a guide RNA targeting human CXCR4 (e.g., human CXCR4 coding exon 2) may include a DNA targeting segment that is at least 90% or at least 95% identical to at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides of the sequence (DNA targeting segment) described in any one of sequence numbers 132-146. Alternatively, a guide RNA targeting human CXCR4 (e.g., human CXCR4 coding exon 2) may include a DNA targeting segment that contains, is essentially derived from, or consists of a sequence that differs from the sequence (DNA targeting segment) described in any one of sequence numbers 132-146 by three or fewer, two or fewer, or one or fewer nucleotides. Alternatively, a guide RNA targeting human CXCR4 (e.g., human CXCR4 coding exon 2) may include a DNA targeting segment that contains, is essentially derived from, or consists of a sequence that differs by three or fewer nucleotides from at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides of the sequence (DNA targeting segment) described in any one of sequence numbers 132-146, by three or fewer nucleotides, two or fewer, or one or fewer nucleotides. In some cases, two or more guide RNAs targeting the target genomic locus (e.g., CXCR4 or human CXCR4) are used.

[0266] TracrRNAs can be in any form (e.g., full-length tracrRNA or active partial tracrRNA) and of various lengths. They can include primary transcripts or processed forms. For example, a tracrRNA (as part of a single guide RNA or as a separate molecule as part of two gRNA molecules) may contain, essentially be, or be part of, all or part of a wild-type tracrRNA sequence (e.g., about 20 or more, about 26 or more, about 32 or more, about 45 or more, about 48 or more, about 54 or more, about 63 or more, about 67 or more, about 85 or more, or even more nucleotides than the wild-type tracrRNA sequence). Examples of wild-type tracrRNA sequences derived from S. pyogenes include versions with 171 nucleotides, 89 nucleotides, 75 nucleotides, and 65 nucleotides. For example, see Deltcheva et al. (2011) Nature 471(7340):602-607, WO2014 / 093661, each of which is incorporated herein by reference in its entirety for all purposes. Examples of tracrRNA within a single guide RNA (sgRNA) include tracrRNA segments found within the +48, ​​+54, +67, and +85 versions of sgRNA, where "+n" indicates that the sgRNA contains up to +n wild-type tracrRNA nucleotides. See U.S. Patent No. 8,697,359, which is incorporated herein by reference in its entirety for all purposes.

[0267] The complementarity percentage between the guide RNA's DNA targeting segment and the complementary strand of the target DNA can be at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%). The complementarity percentage between the DNA targeting segment and the complementary strand of the target DNA can be at least 60% over approximately 20 consecutive nucleotides. As an example, the complementarity percentage between the DNA targeting segment and the complementary strand of the target DNA can be 100% over 14 consecutive nucleotides at the 5' end of the complementary strand of the target DNA, and as low as 0% over the remainder. In such a case, the DNA targeting segment can be considered to be 14 nucleotides long. As another example, the complementarity percentage between the DNA targeting segment and the complementary strand of the target DNA can be 100% over 7 consecutive nucleotides at the 5' end of the complementary strand of the target DNA, and as low as 0% over the remainder. In such cases, the DNA targeting segment can be considered to be 7 nucleotides long. In some guide RNAs, at least 17 nucleotides within the DNA targeting segment are complementary to the complementary strand of the target DNA. For example, the DNA targeting segment may be 20 nucleotides long and may contain one, two, or three mismatches with the complementary strand of the target DNA. In one example, the mismatch is not adjacent to the region of the complementary strand corresponding to the protospacer adjacency motif (PAM) sequence (i.e., the reverse complement of the PAM sequence) (for example, the mismatch is at the 5' end of the DNA targeting segment of the guide RNA, or the mismatch is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 base pairs away from the region of the complementary strand corresponding to the PAM sequence).

[0268] The protein-binding segment of a gRNA may contain two complementary nucleotide sequences. These complementary nucleotides hybridize to form a double-stranded RNA (dsRNA). The protein-binding segment of the target gRNA interacts with a Cas protein, and the gRNA directs the bound Cas protein to a specific nucleotide sequence within the target DNA via its DNA-targeting segment.

[0269] A single guide RNA may contain a DNA targeting segment and a scaffold sequence (i.e., a protein-binding or Cas-binding sequence of the guide RNA). For example, such a guide RNA may have a 5' DNA targeting segment bound to a 3' scaffold sequence. An example scaffold sequence (e.g., for use with S. pyogenes Cas9) is: [ka] [ka] It includes, is essentially derived from, or consists of. In some guide sgRNAs, the four terminal U residues of version 6 are absent. In some sgRNAs, only one, two, or three of the four terminal U residues of version 6 are present. A guide RNA targeting any of the guide RNA target sequences disclosed herein may include, for example, a DNA targeting segment at the 5' end of the guide RNA fused to one of the exemplary guide RNA scaffold sequences at the 3' end of the guide RNA. That is, one of the DNA targeting segments disclosed herein may be bound to the 5' end of any one of the above scaffold sequences to form a single guide RNA (chimeric guide RNA).

[0270] Guide RNA may contain modifications or sequences that provide additional desirable properties (e.g., modified or regulated stability, intracellular targeting, tracking by fluorescent labeling, or protein or protein complex binding sites). That is, guide RNA may contain one or more modified nucleosides or nucleotides, or one or more non-naturally occurring and / or naturally occurring components or configurations used in place of or in addition to canonical A, G, C, and U residues. Examples of such modifications include, for example, 5' caps (e.g., 7-met...

Claims

1. A method for improving the engraftment of donor cells in subjects who require it, (a) Providing donor cells modified to express a first isoform of CXCR4 different from a second isoform of C-X-C chemokine receptor type 4 (CXCR4), wherein the second isoform is expressed in the host cells of the subject; (b) The step of administering the donor cells to the subject, (c) A step of selectively inhibiting host cells in the subject based on the expression of the second isoform of CXCR4, thereby improving the engraftment of donor cells in the subject. Methods that include...

2. The method according to claim 1, wherein the selective inhibition of host cells in step (c) does not involve ablation of host cells by an active killing mechanism.

3. The method according to claim 1 or 2, wherein the selective inhibition in step (c) comprises selectively depleting host cells from the bone marrow.

4. The method according to any one of the preceding claims, wherein the first isoform and the second isoform are functionally indistinguishable but immunologically distinguishable.

5. The method according to any one of the preceding claims, wherein the donor cells express both the first isoform of CXCR4 and the second isoform of CXCR4.

6. The method according to any one of claims 1 to 4, wherein the donor cells express only the first isoform of CXCR4.

7. The method according to either of the preceding claims, wherein the first isoform of CXCR4 is expressed in the donor cells from an expression vector, or a genomic locus is edited to express the first isoform of CXCR4 in the donor cells.

8. The method according to claim 7, wherein the genomic locus is the endogenous CXCR4 genomic locus.

9. The method according to claim 7, wherein the genomic locus is not the endogenous CXCR4 genomic locus.

10. The method according to any of the preceding claims, wherein the selective inhibition in step (c) comprises administering a CXCR4 antagonist to the subject, wherein the CXCR4 antagonist specifically binds to the second isoform of CXCR4 but not to the first isoform of CXCR4, and optionally, step (c) comprises multiple administrations of the CXCR4 antagonist.

11. The method according to claim 10, wherein the CXCR4 antagonist is an antigen-binding protein.

12. The method according to claim 11, wherein the antigen-binding protein is an antibody or an antigen-binding fragment thereof.

13. The antigen-binding protein comprises an immunoglobulin light chain containing three light chain CDRs or its variable region and an immunoglobulin heavy chain containing three heavy chain CDRs or its variable region, Each of the three light chain CDRs contains, essentially consists of, or comprises a sequence that is at least 90% identical to the sequences described in Sequence IDs 15, 17, and 19. The three heavy chain CDRs each contain, essentially consist of, or comprise a sequence that is at least 90% identical to the sequences described in Sequence IDs 7, 9, and 11. The method according to claim 11 or 12.

14. The three light chain CDRs each contain, essentially consist of, or comprise the sequences described in Sequence IDs 15, 17, and 19, The three heavy chain CDRs each contain, essentially consist of, or comprise the sequences described in Sequence IDs 7, 9, and 11. The method according to claim 13.

15. The antigen-binding protein includes, is essentially, or comprises an immunoglobulin light chain variable region that is at least 90% identical to the sequence described in Sequence ID No.

13. The antigen-binding protein includes, is essentially, or comprises an immunoglobulin heavy chain variable region that is at least 90% identical to the sequence described in Sequence ID No.

5. The method according to claim 11 or 12.

16. The immunoglobulin light chain variable region includes, is essentially, or is composed of the sequence described in Sequence ID No.

13. The immunoglobulin heavy chain variable region includes, is essentially, or consists of the sequence described in Sequence ID No.

5. The method according to claim 15.

17. The antigen-binding protein comprises an immunoglobulin light chain containing three light chain CDRs or its variable region and an immunoglobulin heavy chain containing three heavy chain CDRs or its variable region, Each of the three light chain CDRs contains, essentially consists of, or comprises a sequence that is at least 90% identical to the sequences described in Sequence IDs 31, 33, and 35. The three heavy chain CDRs each contain, essentially consist of, or comprise a sequence that is at least 90% identical to the sequences described in SEQ ID NOs: 23, 25, and 27. The method according to claim 11 or 12.

18. The three light chain CDRs each contain, essentially consist of, or comprise the sequences described in sequence numbers 31, 33, and 35, respectively. The three heavy chain CDRs each contain, are essentially, or consist of the sequences described in Sequence IDs 23, 25, and 27. The method according to claim 17.

19. The antigen-binding protein includes, is essentially, or comprises an immunoglobulin light chain variable region that is at least 90% identical to the sequence described in Sequence ID No.

29. The antigen-binding protein includes, is essentially, or comprises an immunoglobulin heavy chain variable region that is at least 90% identical to the sequence described in SEQ ID NO:

21. The method according to claim 11 or 12.

20. The immunoglobulin light chain variable region includes, is essentially, or is composed of the sequence described in Sequence ID No.

29. The immunoglobulin heavy chain variable region includes, is essentially, or consists of the sequence described in Sequence ID No.

21. The method according to claim 19.

21. The method according to any of the preceding claims, wherein the first isoform of CXCR4 is a genetically modified isoform of CXCR4.

22. The method according to any of the preceding claims, wherein the first isoform of CXCR4 is genetically engineered to include a mutation that provides an altered epitope, and optionally the mutation is an artificial mutation.

23. The method according to any one of claims 10 to 20, wherein the first isoform of CXCR4 is genetically engineered to include a mutation providing an altered epitope, optionally the mutation being an artificial mutation, and the altered epitope is located in the binding region of the CXCR4 antagonist, resulting in the CXCR4 antagonist exhibiting reduced or lost ability to bind to and / or inhibit the first isoform of CXCR4 compared to its ability to bind to and / or inhibit the second isoform of CXCR4.

24. The method according to claim 23, wherein both the first isoform of CXCR4 and the second isoform of CXCR4 retain the ability to bind to an endogenous ligand, and the CXCR4 antagonist, if necessary, blocks the binding of the endogenous ligand to the second isoform of CXCR4 but does not block the binding of CXCR4 to the first isoform.

25. The method according to any one of claims 22 to 24, wherein the mutation is located in the extracellular loop 2 (ECL2) region of CXCR4.

26. The method according to any one of claims 22 to 25, wherein the mutation includes an insertion, deletion, or substitution within the region of CXCR4 from position S178 to R183 and / or within the region of R188 to L194.

27. The method according to any one of claims 22 to 26, wherein the mutation comprises one or more mutations selected from F189A, N192A, D193A, S178_E179insK, S178_E179insY, S178_E179insR, E179R, D181R, and D182R, and optionally the mutation comprises (1) F189A, N192A, and D193A, (2) S178_E179insK, (3) S178_E179insY, (4) S178_E179insR, or (5) E179R, D181R, and D182R.

28. The method according to either of the prior claims, wherein steps (b) and (c) are performed simultaneously.

29. (I) Step (b) is performed before step (c), and if necessary, step (c) includes multiple doses of a CXCR4 antagonist after step (b). (II) Step (b) is performed after step (c), and if necessary, step (c) includes multiple doses of a CXCR4 antagonist before step (b), or (III) Step (c) is performed both before and after step (b), and if necessary, step (c) includes multiple doses of the CXCR4 antagonist before step (b) and / or multiple doses of the CXCR4 antagonist after step (b). The method according to any one of claims 1 to 27.

30. A method for improving the engraftment of donor cells in subjects who require it, (a) Providing donor cells that have been modified to express a first isoform of CXCR4 different from a second isoform of CXCR4, wherein the second isoform is expressed in the host cells of the subject; (b) The step of administering the donor cells to the subject, (c) A step of providing the target with means for specifically binding to the second isoform of CXCR4 but not specifically binding to the first isoform of CXCR4. Methods that include...

31. The method according to claim 30, wherein the means for specifically binding to the second isoform of CXCR4 but not specifically binding to the first isoform of CXCR4 selectively inhibits host cells in the subject based on the expression of the second isoform of CXCR4.

32. The method according to claim 31, wherein the selective inhibition of host cells does not involve ablation of host cells by an active killing mechanism.

33. The method according to claim 31 or 32, wherein the selective inhibition of host cells comprises selectively depleting host cells from the bone marrow.

34. The method according to any one of claims 30 to 33, wherein the first isoform and the second isoform are functionally indistinguishable but immunologically distinguishable.

35. The method according to any one of claims 30 to 34, wherein the donor cells express both the first isoform of CXCR4 and the second isoform of CXCR4.

36. The method according to any one of claims 30 to 34, wherein the donor cells express only the first isoform of CXCR4.

37. The method according to any one of claims 30 to 36, wherein the first isoform of CXCR4 is expressed in the donor cells from an expression vector, or a genomic locus is edited to express the first isoform of CXCR4 in the donor cells.

38. The method according to claim 37, wherein the genomic locus is the endogenous CXCR4 genomic locus.

39. The method according to claim 37, wherein the genomic locus is not the endogenous CXCR4 genomic locus.

40. The method according to any one of claims 30 to 39, wherein the first isoform of CXCR4 is a genetically modified isoform of CXCR4.

41. The method according to any one of claims 30 to 40, wherein the first isoform of CXCR4 is genetically engineered to include a mutation that provides an altered epitope, and optionally the mutation is an artificial mutation.

42. The method according to claim 41, wherein the modified epitope is located in the binding region of the means for specifically binding to the second isoform of CXCR4 but not to the first isoform of CXCR4, and as a result, the means for specifically binding to the second isoform of CXCR4 but not to the first isoform of CXCR4 exhibits a reduction or loss of ability to bind to and / or inhibit the first isoform of CXCR4 compared to its ability to bind to and / or inhibit the second isoform of CXCR4.

43. The method according to claim 42, wherein both the first isoform of CXCR4 and the second isoform of CXCR4 retain the ability to bind to an endogenous ligand, and, if necessary, specifically bind to the second isoform of CXCR4 but not specifically bind to the first isoform of CXCR4, wherein the means for blocking the binding of the endogenous ligand to the second isoform of CXCR4 but not to the first isoform of CXCR4.

44. The method according to any one of claims 41 to 43, wherein the mutation is located in the extracellular loop 2 (ECL2) region of CXCR4.

45. The mutation is an insertion, deletion, or substitution within the region of CXCR4 from position S178 to R183 and / or within the region of R188 to L194, according to any one of claims 41 to 44.

46. The method according to any one of claims 41 to 45, wherein the mutation comprises one or more mutations selected from F189A, N192A, D193A, S178_E179insK, S178_E179insY, S178_E179insR, E179R, D181R, and D182R, and optionally the mutation comprises (1) F189A, N192A, and D193A, (2) S178_E179insK, (3) S178_E179insY, (4) S178_E179insR, or (5) E179R, D181R, and D182R.

47. The method according to any one of claims 30 to 46, wherein step (c) includes multiple administrations of the means for which the means specifically binds to the second isoform of CXCR4 but does not specifically bind to the first isoform of CXCR4.

48. The method according to any one of claims 30 to 47, wherein steps (b) and (c) are performed simultaneously.

49. (I) Step (b) is performed before step (c), and if necessary, step (c) is performed after step (b), including multiple doses of the means to specifically bind to the second isoform of CXCR4 but not to the first isoform of CXCR4. (II) Step (b) is performed after step (c), and optionally step (c) is performed before step (b), including multiple doses of the means to specifically bind to the second isoform of CXCR4 but not to the first isoform of CXCR4, or (III) Step (c) is performed both before and after step (b), and if necessary, step (c) includes multiple doses of the means for which the means specifically binds to the second isoform of CXCR4 but not to the first isoform of CXCR4 before step (b) and / or multiple doses of the means for which the means specifically binds to the second isoform of CXCR4 but not to the first isoform of CXCR4 after step (b), The method according to any one of claims 30 to 47.

50. (I) The donor cells and / or host cells are hematopoietic cells, and if necessary, the donor cells and / or host cells are immune cells. (II) The donor cells and / or the host cells are lymphocytes or lymphoid progenitor cells, (III) The donor cells and / or the host cells are T cells, (IV) The donor cells and / or the host cells are tumor-infiltrating lymphocytes (TILs), (V) The donor cells and / or the host cells are B cells, and if necessary, the donor cells and / or the host cells are immature B cells, and the method depletes the host's mature B cells from the bone marrow. (VI) The donor cells and / or the host cells are NK cells, (VII) The donor cells and / or the host cells are hematopoietic stem and progenitor cells, (VIII) The donor cells and / or the host cells are derived from hematopoietic stem cells or hematopoietic stem and progenitor cells, (IX) The donor cells are derived from induced pluripotent stem cells. The method according to any of the prior claims.

51. The method according to any one of the preceding claims, wherein the subject is a mammal or a non-human mammal, and the donor cells are mammalian cells or non-human mammalian cells.

52. The method according to any one of the preceding claims, wherein the subject is a human and the donor cells are human cells.

53. The method according to any of the preceding claims, wherein the donor cells contain or express a therapeutic molecule.

54. The method according to claim 53, wherein the therapeutic molecule does not target CXCR4.

55. The method according to any one of the preceding claims, wherein the donor cells contain or express immunoglobulin, chimeric antigen receptor (CAR), or exogenous T cell receptor (TCR).

56. The method according to claim 55, wherein the immunoglobulin, the CAR, or the exogenous TCR does not target CXCR4.

57. The method according to any of the preceding claims, wherein the donor cells are autologous.

58. The method according to any one of claims 1 to 56, wherein the donor cells are of the same species or lineage.

59. The method according to any prior claim, wherein the subject has a disease or disorder, and the method is for treating the disease or disorder in the subject.

60. The method according to any one of the preceding claims, wherein the subject has cancer, and optionally the cancer is a solid tumor or a hematological cancer.

61. The method according to any prior claim, wherein the subject has a hematopoietic malignancy, and the method is for treating the hematopoietic malignancy in the subject.

62. The method according to any one of the preceding claims, wherein the subject has a deficient immune cell or hereditary hematopoietic failure, and optionally the hereditary hematopoietic failure is sickle cell disease or severe combined immunodeficiency (SCID).

63. The method according to any prior claim, further comprising the step of generating the donor cells by modifying a population of cells to express the first isoform of CXCR4 prior to step (a).

64. (I) The population of cells is a population of induced pluripotent stem cells, and the method further comprises, prior to step (a), a step of differentiating the induced pluripotent stem cells into the donor cells administered in step (a), and, if necessary, differentiating the induced pluripotent stem cells into hematopoietic cells, lymphocytes or lymphoid progenitor cells, T cells, B cells, NK cells, hematopoietic stem cells, or hematopoietic stem and progenitor cells, or (II) The population of cells is a population of hematopoietic stem cells or hematopoietic stem and progenitor cells, and the method further comprises, prior to step (a), a step of differentiating the hematopoietic stem cells or hematopoietic stem and progenitor cells into the donor cells administered in step (a), and, if necessary, differentiating the hematopoietic stem cells or hematopoietic stem and progenitor cells into differentiated hematopoietic cells, lymphocytes or lymphoid progenitor cells, T cells, B cells, or NK cells. The method according to claim 63.

65. The method according to claim 63 or 64, wherein the step of generating the donor cells comprises, prior to step (a), introducing an expression vector encoding the first isoform of CXCR4 to express the first isoform of CXCR4, or the step of generating the donor cells comprises, prior to step (a), editing a genomic locus in the population of cells to express the first isoform of CXCR4.

66. The method according to claim 65, wherein the genomic locus is the endogenous CXCR4 genomic locus.

67. The method according to claim 65, wherein the genomic locus is not the endogenous CXCR4 genomic locus.

68. The aforementioned editing step is, (1) A nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target sequence in the genomic locus, and (2) Exogenous donor nucleic acids, This includes introducing the cell into the aforementioned population of cells. The nuclease agent cleaves the genomic locus, and the exogenous donor nucleic acid is inserted into or recombined with the genomic locus to generate donor cells expressing the first isoform of CXCR4. The method according to any one of claims 65 to 67.

69. The aforementioned nuclease agent, (a) Zinc finger nuclease (ZFN), (b) Transcription activator-like effector nuclease (TALEN), or (c)(i) Cas protein, and (ii) A guide RNA comprising a DNA targeting segment that targets the guide RNA target sequence which is the nuclease target sequence, wherein the guide RNA binds to the Cas protein and directs the Cas protein toward the guide RNA target sequence. The method according to claim 68, including the method described in claim 68.

70. The method according to claim 69, wherein the nuclease agent comprises the Cas protein and the guide RNA, and optionally the DNA targeting segment comprises the sequence described in any one of SEQ ID NOs: 132 to 146, or optionally the guide RNA target sequence comprises the sequence described in any one of SEQ ID NOs: 117 to 131.

71. The method according to claim 69 or 70, wherein the Cas protein is a Cas9 protein.

72. The method according to any one of claims 68 to 71, wherein the exogenous donor nucleic acid includes a homology arm.

73. The method according to any one of claims 68 to 72, wherein the exogenous donor nucleic acid is a single-stranded oligodeoxynucleotide (ssODN).

74. The method according to any one of claims 63 to 73, further comprising the step of isolating the population of cells from the subject or from a different subject before the step of modifying the population of cells.

75. A combination pharmaceutical for administration to subjects who require it, (a) A population of donor cells modified to express a first isoform of CXCR4 that is different from a second isoform of C-X-C chemokine receptor 4 (CXCR4), (b) A CXCR4 antagonist that specifically binds to the second isoform of CXCR4 but does not specifically bind to the first isoform of CXCR4. Combination medicines, including

76. The combination pharmaceutical according to claim 75, wherein the CXCR4 antagonist selectively inhibits host cells in the subject based on the expression of the second isoform of CXCR4.

77. The combination pharmaceutical according to claim 76, wherein the selective inhibition of host cells does not involve ablation of host cells by an active killing mechanism.

78. The combination drug according to claim 76 or 77, wherein the selective inhibition of host cells comprises selectively depleting host cells from the bone marrow.

79. The combination pharmaceutical according to any one of claims 75 to 78, wherein the first isoform and the second isoform are functionally indistinguishable but immunologically distinguishable.

80. The combination pharmaceutical according to any one of claims 75 to 79, wherein the donor cells express both the first isoform of CXCR4 and the second isoform of CXCR4.

81. The combination pharmaceutical according to any one of claims 75 to 79, wherein the donor cells express only the first isoform of CXCR4.

82. The combination pharmaceutical according to any one of claims 75 to 81, wherein the first isoform of CXCR4 is expressed from an expression vector in the donor cell population, or a genomic locus is edited to express the first isoform of CXCR4 in the donor cell population.

83. The combination drug according to claim 82, wherein the genomic locus is the endogenous CXCR4 genomic locus.

84. The combination drug according to claim 82, wherein the genomic locus is not the endogenous CXCR4 genomic locus.

85. The combination pharmaceutical according to any one of claims 75 to 84, wherein the first isoform of CXCR4 is a genetically modified isoform of CXCR4.

86. The combination pharmaceutical according to any one of claims 75 to 85, wherein the first isoform of CXCR4 is genetically engineered to include a mutation that provides an altered epitope, and optionally the mutation is an artificial mutation.

87. The combination pharmacopoe according to claim 86, wherein the modified epitope is located in the binding region of the CXCR4 antagonist, and as a result, the CXCR4 antagonist exhibits a reduced or lost ability to bind to and / or inhibit the first isoform of CXCR4 compared to its ability to bind to and / or inhibit the second isoform of CXCR4.

88. The combination pharmaceutical according to claim 87, wherein both the first isoform of CXCR4 and the second isoform of CXCR4 retain the ability to bind to an endogenous ligand, and the CXCR4 antagonist, if necessary, blocks the binding of the endogenous ligand to the second isoform of CXCR4 but does not block the binding of CXCR4 to the first isoform.

89. The combination pharmaceutical according to any one of claims 86 to 88, wherein the mutation is located in the extracellular loop 2 (ECL2) region of CXCR4.

90. The combination pharmaceutical according to any one of claims 86 to 89, wherein the mutation includes an insertion, deletion, or substitution within the region of CXCR4 from position S178 to R183 and / or within the region of R188 to L194.

91. The combination pharmaceutical according to any one of claims 86 to 90, wherein the mutation comprises one or more mutations selected from F189A, N192A, D193A, S178_E179insK, S178_E179insY, S178_E179insR, E179R, D181R, and D182R, and optionally the mutation comprises (1) F189A, N192A, and D193A, (2) S178_E179insK, (3) S178_E179insY, (4) S178_E179insR, or (5) E179R, D181R, and D182R.

92. The combination pharmaceutical according to any one of claims 75 to 91, wherein the CXCR4 antagonist is an antigen-binding protein.

93. The combination pharmaceutical according to claim 92, wherein the antigen-binding protein is an antibody or an antigen-binding fragment thereof.

94. The antigen-binding protein comprises an immunoglobulin light chain containing three light chain CDRs or its variable region and an immunoglobulin heavy chain containing three heavy chain CDRs or its variable region, Each of the three light chain CDRs contains, essentially consists of, or comprises a sequence that is at least 90% identical to the sequences described in Sequence IDs 15, 17, and 19. The three heavy chain CDRs each contain, essentially consist of, or comprise a sequence that is at least 90% identical to the sequences described in Sequence IDs 7, 9, and 11. The combination pharmaceutical product according to claim 92 or 93.

95. The three light chain CDRs each contain, essentially consist of, or comprise the sequences described in Sequence IDs 15, 17, and 19, The three heavy chain CDRs each contain, essentially consist of, or comprise the sequences described in Sequence IDs 7, 9, and 11. The combination drug according to claim 94.

96. The antigen-binding protein includes, is essentially, or comprises an immunoglobulin light chain variable region that is at least 90% identical to the sequence described in Sequence ID No.

13. The antigen-binding protein includes, is essentially, or comprises an immunoglobulin heavy chain variable region that is at least 90% identical to the sequence described in Sequence ID No.

5. The combination pharmaceutical product according to claim 92 or 93.

97. The immunoglobulin light chain variable region includes, is essentially, or is composed of the sequence described in Sequence ID No.

13. The immunoglobulin heavy chain variable region includes, is essentially, or consists of the sequence described in Sequence ID No.

5. The combination pharmaceutical product according to claim 96.

98. The antigen-binding protein comprises an immunoglobulin light chain containing three light chain CDRs or its variable region and an immunoglobulin heavy chain containing three heavy chain CDRs or its variable region, Each of the three light chain CDRs contains, essentially consists of, or comprises a sequence that is at least 90% identical to the sequences described in Sequence IDs 31, 33, and 35. The three heavy chain CDRs each contain, essentially consist of, or comprise a sequence that is at least 90% identical to the sequences described in SEQ ID NOs: 23, 25, and 27. The combination pharmaceutical product according to claim 92 or 93.

99. The three light chain CDRs each contain, essentially consist of, or comprise the sequences described in sequence numbers 31, 33, and 35, respectively. The three heavy chain CDRs each contain, are essentially, or consist of the sequences described in Sequence IDs 23, 25, and 27. The combination pharmaceutical product according to claim 98.

100. The antigen-binding protein includes, is essentially, or comprises an immunoglobulin light chain variable region that is at least 90% identical to the sequence described in Sequence ID No.

29. The antigen-binding protein includes, is essentially, or comprises an immunoglobulin heavy chain variable region that is at least 90% identical to the sequence described in SEQ ID NO:

21. The combination pharmaceutical product according to claim 92 or 93.

101. The immunoglobulin light chain variable region includes, is essentially, or is composed of the sequence described in Sequence ID No.

29. The immunoglobulin heavy chain variable region includes, is essentially, or consists of the sequence described in Sequence ID No.

21. The combination pharmaceutical product according to claim 100.

102. A combination pharmaceutical for administration to subjects who require it, (a) A population of donor cells modified to express a first isoform of CXCR4 that is different from a second isoform of CXCR4, (b) A combination pharmaceutical comprising means for specifically binding to the second isoform of CXCR4 but not specifically binding to the first isoform of CXCR4.

103. The combination pharmaceutical according to claim 102, wherein the means for specifically binding to the second isoform of CXCR4 but not specifically binding to the first isoform of CXCR4 selectively inhibits host cells in the target based on the expression of the second isoform of CXCR4.

104. The combination pharmaceutical according to claim 103, wherein the selective inhibition of host cells does not involve ablation of host cells by an active killing mechanism.

105. The combination drug according to claim 103 or 104, wherein the selective inhibition of host cells comprises selectively depleting host cells from the bone marrow.

106. The combination pharmaceutical according to any one of claims 102 to 105, wherein the first isoform and the second isoform are functionally indistinguishable but immunologically distinguishable.

107. The combination pharmaceutical according to any one of claims 102 to 106, wherein the donor cells express both the first isoform of CXCR4 and the second isoform of CXCR4.

108. The combination pharmaceutical according to any one of claims 102 to 106, wherein the donor cells express only the first isoform of CXCR4.

109. The combination pharmaceutical according to any one of claims 102 to 108, wherein the first isoform of CXCR4 is expressed from an expression vector in the donor cell population, or a genomic locus is edited to express the first isoform of CXCR4 in the donor cell population.

110. The combination drug according to claim 109, wherein the genomic locus is the endogenous CXCR4 genomic locus.

111. The combination drug according to claim 109, wherein the genomic locus is not the endogenous CXCR4 genomic locus.

112. The combination pharmaceutical according to any one of claims 102 to 111, wherein the first isoform of CXCR4 is a genetically modified isoform of CXCR4.

113. The combination pharmaceutical according to any one of claims 102 to 112, wherein the first isoform of CXCR4 is genetically engineered to include a mutation that provides an altered epitope, and optionally the mutation is an artificial mutation.

114. The combination pharmaceutical according to claim 113, wherein the modified epitope is located in the binding region of the means for specifically binding to the second isoform of CXCR4 but not to the first isoform of CXCR4, and as a result, the means for specifically binding to the second isoform of CXCR4 but not to the first isoform of CXCR4 exhibits a reduction or loss of ability to bind to and / or inhibit the first isoform of CXCR4 compared to its ability to bind to and / or inhibit the second isoform of CXCR4.

115. The combination pharmaceutical according to claim 114, wherein both the first isoform of CXCR4 and the second isoform of CXCR4 retain the ability to bind to an endogenous ligand, and, if necessary, specifically bind to the second isoform of CXCR4 but not specifically bind to the first isoform of CXCR4, wherein the means for blocking the binding of the endogenous ligand to the second isoform of CXCR4 but not to the first isoform of CXCR4.

116. The combination pharmaceutical according to any one of claims 113 to 115, wherein the mutation is located in the extracellular loop 2 (ECL2) region of CXCR4.

117. The combination pharmaceutical according to any one of claims 113 to 116, wherein the mutation includes an insertion, deletion, or substitution within the region of CXCR4 from position S178 to R183 and / or within the region of R188 to L194.

118. The combination pharmaceutical according to any one of claims 113 to 117, wherein the mutation comprises one or more mutations selected from F189A, N192A, D193A, S178_E179insK, S178_E179insY, S178_E179insR, E179R, D181R, and D182R, and optionally the mutation comprises (1) F189A, N192A, and D193A, (2) S178_E179insK, (3) S178_E179insY, (4) S178_E179insR, or (5) E179R, D181R, and D182R.

119. (I) The donor cells are hematopoietic cells, and if necessary, the donor cells are immune cells. (II) The donor cells are lymphocytes or lymphoid progenitor cells, (III) The donor cells are T cells. (IV) The donor cells are tumor-infiltrating lymphocytes (TILs), (V) The donor cells are B cells, (VI) The donor cells are NK cells. (VII) The donor cells are hematopoietic stem cells or hematopoietic stem and progenitor cells, (VIII) The donor cells are derived from induced pluripotent stem cells, or from hematopoietic stem cells or hematopoietic stem and progenitor cells, A combination pharmaceutical product according to any one of claims 75 to 118.

120. The combination pharmaceutical according to any one of claims 75 to 119, wherein the subject is a mammal or a non-human mammal, and the donor cells are mammalian cells or non-human mammalian cells.

121. The combination pharmaceutical according to any one of claims 75 to 120, wherein the subject is a human and the donor cells are human cells.

122. The combination pharmaceutical according to any one of claims 75 to 121, wherein the donor cells contain or express a therapeutic molecule.

123. The combination pharmaceutical according to claim 122, wherein the therapeutic molecule does not target CXCR4.

124. The combination pharmaceutical according to any one of claims 75 to 123, wherein the donor cells contain or express immunoglobulin, chimeric antigen receptor (CAR), or exogenous T cell receptor (TCR).

125. The combination pharmaceutical according to claim 124, wherein the immunoglobulin, the CAR, or the exogenous TCR does not target CXCR4.

126. The combination pharmaceutical according to any one of claims 75 to 125, wherein the donor cells are autologous.

127. The combination pharmaceutical according to any one of claims 75 to 125, wherein the donor cells are of the same species or of the same lineage.

128. The combination pharmaceutical according to any one of claims 75 to 127, wherein the subject has a disease or disorder, and the combination pharmaceutical is for treating the disease or disorder in the subject.

129. The combination pharmaceutical according to any one of claims 75 to 128, wherein the subject has cancer, and optionally the cancer is a solid tumor or a hematological cancer.

130. The combination pharmaceutical according to any one of claims 75 to 129, wherein the subject has a hematopoietic malignancy, and the combination pharmaceutical is for treating the hematopoietic malignancy in the subject.

131. The combination pharmaceutical according to any one of claims 75 to 130, wherein the subject has a deficient immune cell or hereditary hematopoietic failure, and optionally the hereditary hematopoietic failure is sickle cell disease or severe combined immunodeficiency (SCID).

132. An isolated cell or population of cells modified to express a first isoform of CXCR4 different from a second isoform of CXCR4, wherein the first isoform of CXCR4 is genetically engineered to include a mutation providing an altered epitope, the altered epitope being located in the binding region of a CXCR4 antagonist, resulting in the CXCR4 antagonist exhibiting reduced or lost ability to bind to and / or inhibit the first isoform of CXCR4 compared to its ability to bind to and / or inhibit the second isoform of CXCR4, and the first isoform of CXCR4 retains its binding to its endogenous ligand.

133. The isolated cells or population of cells according to claim 132, wherein the mutation is an artificial mutation.

134. An isolated cell or population of cells according to claim 132 or 133, wherein both the first isoform of CXCR4 and the second isoform of CXCR4 retain the ability to bind to an endogenous ligand, and the CXCR4 antagonist, if necessary, blocks the binding of the endogenous ligand to the second isoform of CXCR4 but does not block the binding of CXCR4 to the first isoform.

135. The isolated cells or population of cells according to any one of claims 132 to 134, wherein the first isoform and the second isoform are functionally indistinguishable but immunologically distinguishable.

136. The isolated cells or population of cells according to any one of claims 132 to 135, wherein the cells (one or more) express both the first isoform of CXCR4 and the second isoform of CXCR4.

137. The isolated cells or population of cells according to any one of claims 132 to 135, wherein the cells (one or more) express only the first isoform of CXCR4.

138. An isolated cell or population of cells according to any one of claims 132 to 137, wherein the first isoform of CXCR4 is expressed in the cell(s)(s) from an expression vector, or a genomic locus is edited to express the first isoform of CXCR4 in the cell(s).

139. The isolated cell or population of cells according to claim 138, wherein the genomic locus is the endogenous CXCR4 genomic locus.

140. The isolated cells or population of cells according to claim 138, wherein the genomic locus is not the endogenous CXCR4 genomic locus.

141. The isolated cell or population of cells according to any one of claims 132 to 140, wherein the mutation is located in the extracellular loop 2 (ECL2) region of CXCR4.

142. An isolated cell or population of cells according to any one of claims 132 to 141, wherein the mutation comprises an insertion, deletion, or substitution within the region of CXCR4 from position S178 to R183 and / or within the region of R188 to L194.

143. The isolated cells or population of cells according to any one of claims 132 to 142, wherein the mutation comprises one or more mutations selected from F189A, N192A, D193A, S178_E179insK, S178_E179insY, S178_E179insR, E179R, D181R, and D182R, and optionally the mutation comprises (1) F189A, N192A, and D193A, (2) S178_E179insK, (3) S178_E179insY, (4) S178_E179insR, or (5) E179R, D181R, and D182R.

144. An isolated cell or population of cells according to any one of claims 132 to 143, wherein the first isoform and the second isoform are immunologically distinguishable by the CXCR4 antagonist, and the CXCR4 antagonist specifically binds to the second isoform of CXCR4 but not specifically binds to the first isoform of CXCR4.

145. The isolated cell or population of cells according to claim 144, wherein the CXCR4 antagonist is an antigen-binding protein.

146. The isolated cells or population of cells according to claim 145, wherein the antigen-binding protein is an antibody or an antigen-binding fragment thereof.

147. The antigen-binding protein comprises an immunoglobulin light chain containing three light chain CDRs or its variable region and an immunoglobulin heavy chain containing three heavy chain CDRs or its variable region, Each of the three light chain CDRs contains, essentially consists of, or comprises a sequence that is at least 90% identical to the sequences described in Sequence IDs 15, 17, and 19. The three heavy chain CDRs each contain, essentially consist of, or comprise a sequence that is at least 90% identical to the sequences described in Sequence IDs 7, 9, and 11. The isolated cells or population of cells according to claim 145 or 146.

148. The three light chain CDRs each contain, essentially consist of, or comprise the sequences described in Sequence IDs 15, 17, and 19, The three heavy chain CDRs each contain, essentially consist of, or comprise the sequences described in Sequence IDs 7, 9, and 11. The isolated cells or population of cells according to claim 147.

149. The antigen-binding protein includes, is essentially, or comprises an immunoglobulin light chain variable region that is at least 90% identical to the sequence described in Sequence ID No.

13. The antigen-binding protein includes, is essentially, or comprises an immunoglobulin heavy chain variable region that is at least 90% identical to the sequence described in Sequence ID No.

5. The isolated cells or population of cells according to claim 145 or 146.

150. The immunoglobulin light chain variable region includes, is essentially, or is composed of the sequence described in Sequence ID No.

13. The immunoglobulin heavy chain variable region includes, is essentially, or consists of the sequence described in Sequence ID No.

5. The isolated cells or population of cells according to claim 149.

151. The antigen-binding protein comprises an immunoglobulin light chain containing three light chain CDRs or its variable region and an immunoglobulin heavy chain containing three heavy chain CDRs or its variable region, Each of the three light chain CDRs contains, essentially consists of, or comprises a sequence that is at least 90% identical to the sequences described in Sequence IDs 31, 33, and 35. The three heavy chain CDRs each contain, essentially consist of, or comprise a sequence that is at least 90% identical to the sequences described in SEQ ID NOs: 23, 25, and 27. The isolated cells or population of cells according to claim 145 or 146.

152. The three light chain CDRs each contain, essentially consist of, or comprise the sequences described in sequence numbers 31, 33, and 35, respectively. The three heavy chain CDRs each contain, are essentially, or consist of the sequences described in Sequence IDs 23, 25, and 27. The isolated cells or population of cells according to claim 151.

153. The antigen-binding protein includes, is essentially, or comprises an immunoglobulin light chain variable region that is at least 90% identical to the sequence described in Sequence ID No.

29. The antigen-binding protein includes, is essentially, or comprises an immunoglobulin heavy chain variable region that is at least 90% identical to the sequence described in SEQ ID NO:

21. The isolated cells or population of cells according to claim 145 or 146.

154. The immunoglobulin light chain variable region includes, is essentially, or is composed of the sequence described in Sequence ID No.

29. The immunoglobulin heavy chain variable region includes, is essentially, or consists of the sequence described in Sequence ID No.

21. The isolated cells or population of cells according to claim 153.

155. (I) The cells (one or more) are hematopoietic cells (may be multiple), and if necessary, the cells (one or more) are immune cells (may be multiple). (II) The cells (one or more) are lymphocytes or lymphoid progenitor cells (may be multiple), (III) The cells (one or more) are T cells (may be multiple). (IV) The cells (one or more) are tumor-infiltrating lymphocytes (TILs) (may be multiple). (V) The cell(s) (one or more) is a B cell(s), (VI) The cells (one or more) are NK cells (may be multiple). (VII) The cells (one or more) are hematopoietic stem cells (may be multiple) or hematopoietic stem and progenitor cells (may be multiple), (VIII) The cells (one or more) are induced pluripotent stem cells (may be multiple). An isolated cell or population of cells according to any one of claims 132 to 154.

156. The isolated cells or population of cells according to any one of claims 132 to 155, wherein the cells (one or more) are mammalian cells (majority) or non-human mammalian cells (majority).

157. The isolated cells or population of cells according to any one of claims 132 to 156, wherein the cells (one or more) are human cells (may be multiple).

158. The isolated cells or population of cells according to any one of claims 132 to 157, wherein the cells (one or more) contain or express a therapeutic molecule.

159. The isolated cells or population of cells according to claim 158, wherein the therapeutic molecule does not target CXCR4.

160. The isolated cells or population of cells according to any one of claims 132 to 159, wherein the cells (one or more) contain or express immunoglobulin, chimeric antigen receptor (CAR), or exogenous T cell receptor (TCR).

161. The isolated cells or population of cells according to claim 160, wherein the immunoglobulin, the CAR, or the exogenous TCR does not target CXCR4.

162. The isolated cells or population of cells according to any one of claims 132 to 161, wherein the cells (one or more) are isolated from the subject.

163. Isolated cells or populations of cells according to any one of claims 132 to 162, for use in the treatment of a subject having cells expressing the second isoform of CXCR4.

164. The isolated cells or population of cells for use according to claim 163, wherein the cells (one or more) are isolated from the subject.

165. A method for preparing an isolated cell or population of cells according to any one of claims 132 to 164, comprising the step of modifying the cell or population of cells to express the first isoform of CXCR4.

166. The method according to claim 165, wherein the modification step includes introducing an expression vector encoding the first isoform of CXCR4, or the modification step includes editing a genomic locus to express the first isoform of CXCR4.

167. The method according to claim 166, wherein the genomic locus is the endogenous CXCR4 genomic locus.

168. The method according to claim 166, wherein the genomic locus is not the endogenous CXCR4 genomic locus.

169. The aforementioned editing step is, (1) A nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target sequence in the genomic locus, and (2) Exogenous donor nucleic acids, This includes introducing the following into the cells: The nuclease agent cleaves the genomic locus, and the exogenous donor nucleic acid is inserted into or recombined with the genomic locus to produce cells expressing the first isoform of CXCR4. The method according to any one of claims 166 to 168.

170. The aforementioned nuclease agent, (a) Zinc finger nuclease (ZFN), (b) Transcription activator-like effector nuclease (TALEN), or (c)(i) Cas protein, and (ii) A guide RNA comprising a DNA targeting segment that targets the guide RNA target sequence which is the nuclease target sequence, wherein the guide RNA binds to the Cas protein and directs the Cas protein toward the guide RNA target sequence. The method according to claim 169, including the method described in claim 169.

171. The method according to claim 170, wherein the nuclease agent comprises the Cas protein and the guide RNA, and optionally the DNA targeting segment comprises the sequence described in any one of SEQ ID NOs: 132 to 146, or optionally the guide RNA target sequence comprises the sequence described in any one of SEQ ID NOs: 117 to 131.

172. The method according to claim 170 or 171, wherein the Cas protein is a Cas9 protein.

173. The method according to any one of claims 169 to 172, wherein the exogenous donor nucleic acid includes a homology arm.

174. The method according to any one of claims 169 to 173, wherein the exogenous donor nucleic acid is a single-stranded oligodeoxynucleotide (ssODN).

175. A genetically engineered human C-X-C chemokine receptor 4 (CXCR4) protein comprising an artificial mutation providing an altered epitope, wherein the altered epitope is located in the binding region of a CXCR4 antagonist, and as a result, the CXCR4 antagonist exhibits reduced or lost ability to bind to and / or inhibit the genetically engineered human CXCR4 protein compared to its ability to bind to and / or inhibit the wild-type human CXCR4 protein, and the genetically engineered human CXCR4 protein retains its binding to its endogenous ligand(s).

176. The genetically modified human CXCR4 protein according to claim 175, wherein the genetically modified CXCR4 protein is functionally indistinguishable from native CXCR4 protein but immunologically distinguishable.

177. The genetically engineered human CXCR4 protein according to claim 175 or 176, wherein the mutation is located in the extracellular loop 2 (ECL2) region of CXCR4.

178. The genetically engineered human CXCR4 protein according to any one of claims 175 to 177, wherein the mutation comprises an insertion, deletion, or substitution within the region of CXCR4 from S178 to R183 and / or within the region of CXCR4 from R188 to L194.

179. The genetically engineered human CXCR4 protein according to any one of claims 175 to 178, wherein the mutation comprises one or more mutations selected from F189A, N192A, D193A, S178_E179insK, S178_E179insY, S178_E179insR, E179R, D181R, and D182R, and optionally the mutation comprises (1) F189A, N192A, and D193A, (2) S178_E179insK, (3) S178_E179insY, (4) S178_E179insR, or (5) E179R, D181R, and D182R.

180. The genetically modified human CXCR4 protein according to any one of claims 175 to 179, wherein the genetically modified CXCR4 protein and the native CXCR4 protein are functionally indistinguishable by the CXCR4 antagonist but immunologically distinguishable.

181. The genetically modified human CXCR4 protein according to claim 180, wherein the CXCR4 antagonist is an antigen-binding protein.

182. The genetically modified human CXCR4 protein according to claim 181, wherein the antigen-binding protein is an antibody or an antigen-binding fragment thereof.

183. The antigen-binding protein comprises an immunoglobulin light chain containing three light chain CDRs or its variable region and an immunoglobulin heavy chain containing three heavy chain CDRs or its variable region, Each of the three light chain CDRs contains, essentially consists of, or comprises a sequence that is at least 90% identical to the sequences described in Sequence IDs 15, 17, and 19. The three heavy chain CDRs each contain, essentially consist of, or comprise a sequence that is at least 90% identical to the sequences described in Sequence IDs 7, 9, and 11. The genetically modified human CXCR4 protein according to claim 181 or 182.

184. The three light chain CDRs each contain, essentially consist of, or comprise the sequences described in Sequence IDs 15, 17, and 19, The three heavy chain CDRs each contain, essentially consist of, or comprise the sequences described in Sequence IDs 7, 9, and 11. The genetically modified human CXCR4 protein according to claim 183.

185. The antigen-binding protein includes, is essentially, or comprises an immunoglobulin light chain variable region that is at least 90% identical to the sequence described in Sequence ID No.

13. The antigen-binding protein includes, is essentially, or comprises an immunoglobulin heavy chain variable region that is at least 90% identical to the sequence described in Sequence ID No.

5. The genetically modified human CXCR4 protein according to claim 181 or 182.

186. The immunoglobulin light chain variable region includes, is essentially, or is composed of the sequence described in Sequence ID No.

13. The immunoglobulin heavy chain variable region includes, is essentially, or consists of the sequence described in Sequence ID No.

5. The genetically modified human CXCR4 protein according to claim 185.

187. The antigen-binding protein comprises an immunoglobulin light chain containing three light chain CDRs or its variable region and an immunoglobulin heavy chain containing three heavy chain CDRs or its variable region, Each of the three light chain CDRs contains, essentially consists of, or comprises a sequence that is at least 90% identical to the sequences described in Sequence IDs 31, 33, and 35. The three heavy chain CDRs each contain, essentially consist of, or comprise a sequence that is at least 90% identical to the sequences described in SEQ ID NOs: 23, 25, and 27. The genetically modified human CXCR4 protein according to claim 181 or 182.

188. The three light chain CDRs each contain, essentially consist of, or comprise the sequences described in sequence numbers 31, 33, and 35, respectively. The three heavy chain CDRs each contain, are essentially, or consist of the sequences described in Sequence IDs 23, 25, and 27. The genetically modified human CXCR4 protein according to claim 187.

189. The antigen-binding protein includes, is essentially, or comprises an immunoglobulin light chain variable region that is at least 90% identical to the sequence described in Sequence ID No.

29. The antigen-binding protein includes, is essentially, or comprises an immunoglobulin heavy chain variable region that is at least 90% identical to the sequence described in SEQ ID NO:

21. The genetically modified human CXCR4 protein according to claim 181 or 182.

190. The immunoglobulin light chain variable region includes, is essentially, or is composed of the sequence described in Sequence ID No.

29. The immunoglobulin heavy chain variable region includes, is essentially, or consists of the sequence described in Sequence ID No.

21. The genetically modified human CXCR4 protein according to claim 189.

191. A nucleic acid encoding a genetically modified human CXCR4 protein according to any one of claims 175 to 190, wherein the nucleic acid is, optionally, an expression vector encoding the genetically modified human CXCR4 protein.

192. A method for producing a genetically engineered human C-X-C chemokine receptor 4 (CXCR4) protein containing an artificial mutation providing an altered epitope, wherein the altered epitope is located in the binding region of a CXCR4 antagonist, and as a result, the CXCR4 antagonist exhibits reduced or lost ability to bind to and / or inhibit the genetically engineered human CXCR4 protein compared to its ability to bind to and / or inhibit the wild-type human CXCR4 protein, and the genetically engineered human CXCR4 protein retains its binding to its endogenous ligand(s), and the method is, (a) The step of determining the epitope in the binding region of the CXCR4 antagonist, (b) the step of selecting a site for generating the artificial mutation that provides the modified epitope in the binding region of the CXCR4 antagonist, (c) A step of generating the genetically engineered human CXCR4 protein, which includes the artificial mutation that provides the modified epitope in the binding region of the CXCR4 antagonist, (d) Testing the genetically modified human CXCR4 protein to determine whether the CXCR4 antagonist exhibits reduced or lost ability to bind to and / or inhibit the genetically modified human CXCR4 protein compared to its ability to bind to and / or inhibit the wild-type human CXCR4 protein, and to determine whether the genetically modified human CXCR4 protein retains binding to its endogenous ligand(s). Methods that include...

193. The method according to claim 192, wherein the epitope of the CXCR4 antagonist is determined by alanine scanning mutation analysis, peptide blot analysis, peptide cleavage analysis, crystallographic study, NMR analysis, epitope excision, epitope extraction, antigen chemical modification, and / or hydrogen / deuterium exchange detected by mass spectrometry.

194. The method according to claim 192 or 193, wherein the epitope of the CXCR4 antagonist is determined by high-resolution cryo-electron microscopy analysis of the CXCR4 antagonist complexed with human CXCR4 protein.

195. The site of the aforementioned artificial mutation is (I) Non-conserved among different mammalian species, (II) Not causing secondary structural changes, (III) Located in a site accessible to ligand binding, (IV) Not located in a site involved in a predicted or experimentally established or confirmed protein-protein interaction, (V) Not resulting in the loss or introduction of disulfide bonds, intermolecular or intramolecular interactions, or hydrophobic stacking. (VI) Not resulting in the deletion or introduction of post-translational protein modification sites, and / or (VII) Located in a region with a unique topology compared to other mammalian proteins, as determined by crystal structure analysis or computer-aided structural prediction. The method according to any one of claims 192 to 194, as selected.

196. The method according to any one of claims 192 to 195, wherein the step of generating the genetically modified human CXCR4 protein comprises modifying a cell or population of cells to express the genetically modified human CXCR4 protein.

197. The method according to claim 196, wherein the modification step comprises introducing an expression vector encoding the genetically modified human CXCR4 protein, or the modification step comprises editing a genomic locus to express the genetically modified human CXCR4 protein.

198. The method according to claim 197, wherein the genomic locus is the endogenous CXCR4 genomic locus.

199. The method according to claim 197, wherein the genomic locus is not the endogenous CXCR4 genomic locus.

200. The aforementioned editing step is, (1) A nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target sequence in the genomic locus, and (2) Exogenous donor nucleic acids, This includes introducing the following into the cells: The nuclease agent cleaves the genomic locus, and the exogenous donor nucleic acid is inserted into or recombined with the genomic locus to generate cells that express the genetically modified human CXCR4 protein. The method according to any one of claims 197 to 199.

201. The aforementioned nuclease agent, (a) Zinc finger nuclease (ZFN), (b) Transcription activator-like effector nuclease (TALEN), or (c)(i) Cas protein, and (ii) A guide RNA comprising a DNA targeting segment that targets the guide RNA target sequence which is the nuclease target sequence, wherein the guide RNA binds to the Cas protein and directs the Cas protein toward the guide RNA target sequence. The method according to claim 200, including the method described in claim 200.

202. The method according to claim 201, wherein the nuclease agent comprises the Cas protein and the guide RNA, and optionally the DNA targeting segment comprises the sequence described in any one of SEQ ID NOs: 132 to 146, or optionally the guide RNA target sequence comprises the sequence described in any one of SEQ ID NOs: 117 to 131.

203. The method according to claim 201 or 202, wherein the Cas protein is a Cas9 protein.

204. The method according to any one of claims 200 to 203, wherein the exogenous donor nucleic acid includes a homology arm.

205. The method according to any one of claims 200 to 204, wherein the exogenous donor nucleic acid is a single-stranded oligodeoxynucleotide (ssODN).

206. The method according to any one of claims 192 to 205, wherein the genetically modified human CXCR4 protein is functionally indistinguishable from native CXCR4 protein but immunologically distinguishable.

207. The method according to any one of claims 192 to 206, wherein the mutation is located in the extracellular loop 2 (ECL2) region of CXCR4.

208. The method according to any one of claims 192 to 207, wherein the mutation includes an insertion, deletion, or substitution within the region of CXCR4 from position S178 to R183 and / or within the region of R188 to L194.

209. The method according to any one of claims 192 to 208, wherein the mutation comprises one or more mutations selected from F189A, N192A, D193A, S178_E179insK, S178_E179insY, S178_E179insR, E179R, D181R, and D182R, and optionally the mutation comprises (1) F189A, N192A, and D193A, (2) S178_E179insK, (3) S178_E179insY, (4) S178_E179insR, or (5) E179R, D181R, and D182R.

210. The method according to any one of claims 192 to 209, wherein the genetically modified human CXCR4 protein and the native CXCR4 protein are functionally indistinguishable by the CXCR4 antagonist but immunologically distinguishable.

211. The method according to any one of claims 192 to 210, wherein the CXCR4 antagonist is an antigen-binding protein.

212. The method according to claim 211, wherein the antigen-binding protein is an antibody or an antigen-binding fragment thereof.

213. The antigen-binding protein comprises an immunoglobulin light chain containing three light chain CDRs or its variable region and an immunoglobulin heavy chain containing three heavy chain CDRs or its variable region, Each of the three light chain CDRs contains, essentially consists of, or comprises a sequence that is at least 90% identical to the sequences described in Sequence IDs 15, 17, and 19. The three heavy chain CDRs each contain, essentially consist of, or comprise a sequence that is at least 90% identical to the sequences described in Sequence IDs 7, 9, and 11. The method according to claim 211 or 212.

214. The three light chain CDRs each contain, essentially consist of, or comprise the sequences described in Sequence IDs 15, 17, and 19, The three heavy chain CDRs each contain, essentially consist of, or comprise the sequences described in Sequence IDs 7, 9, and 11. The method according to claim 213.

215. The antigen-binding protein includes, is essentially, or comprises an immunoglobulin light chain variable region that is at least 90% identical to the sequence described in Sequence ID No.

13. The antigen-binding protein includes, is essentially, or comprises an immunoglobulin heavy chain variable region that is at least 90% identical to the sequence described in Sequence ID No.

5. The method according to claim 211 or 212.

216. The immunoglobulin light chain variable region includes, is essentially, or is composed of the sequence described in Sequence ID No.

13. The immunoglobulin heavy chain variable region includes, is essentially, or consists of the sequence described in Sequence ID No.

5. The method according to claim 215.

217. The antigen-binding protein comprises an immunoglobulin light chain containing three light chain CDRs or its variable region and an immunoglobulin heavy chain containing three heavy chain CDRs or its variable region, Each of the three light chain CDRs contains, essentially consists of, or comprises a sequence that is at least 90% identical to the sequences described in Sequence IDs 31, 33, and 35. The three heavy chain CDRs each contain, essentially consist of, or comprise a sequence that is at least 90% identical to the sequences described in SEQ ID NOs: 23, 25, and 27. The method according to claim 211 or 212.

218. The three light chain CDRs each contain, essentially consist of, or comprise the sequences described in sequence numbers 31, 33, and 35, respectively. The three heavy chain CDRs each contain, are essentially, or consist of the sequences described in Sequence IDs 23, 25, and 27. The method according to claim 217.

219. The antigen-binding protein includes, is essentially, or comprises an immunoglobulin light chain variable region that is at least 90% identical to the sequence described in Sequence ID No.

29. The antigen-binding protein includes, is essentially, or comprises an immunoglobulin heavy chain variable region that is at least 90% identical to the sequence described in SEQ ID NO:

21. The method according to claim 211 or 212.

220. The immunoglobulin light chain variable region includes, is essentially, or is composed of the sequence described in Sequence ID No.

29. The immunoglobulin heavy chain variable region includes, is essentially, or consists of the sequence described in Sequence ID No.

21. The method according to claim 219.

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