CD64 CHIMERIC RECEPTOR AND USES THEREOF
20240082400 ยท 2024-03-14
Inventors
Cpc classification
C07K16/2863
CHEMISTRY; METALLURGY
A61K39/4611
HUMAN NECESSITIES
C07K2319/30
CHEMISTRY; METALLURGY
A61K2039/507
HUMAN NECESSITIES
C07K2317/24
CHEMISTRY; METALLURGY
A61K39/3955
HUMAN NECESSITIES
C07K14/70535
CHEMISTRY; METALLURGY
C07K14/70596
CHEMISTRY; METALLURGY
A61K2239/38
HUMAN NECESSITIES
A61K39/464402
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
A61K39/00
HUMAN NECESSITIES
C07K14/705
CHEMISTRY; METALLURGY
A61K39/395
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
Abstract
The present invention provides compositions and methods for treating cancer in a human. The invention includes a chimeric receptor which comprises an CD64 binding domain, a transmembrane domain, and a CD3zeta signaling domain.
Claims
1. A chimeric receptor comprising a.) an extracellular ligand binding domain of CD64; b.) a transmembrane domain; and c.) an intracellular domain.
2. The chimeric receptor of claim 1, further comprising a signal peptide.
3. The chimeric receptor of claim 2, wherein the signal peptide is a mouse CD64 signal peptide.
4. The chimeric receptor of claim 3, wherein the mouse CD64 signal peptide is humanized.
5. The chimeric receptor of claim 1, wherein the extracellular ligand binding domain comprises D1, D2, and D3 of CD64.
6. The chimeric receptor of claim 1, wherein the transmembrane domain comprises a CD64 transmembrane domain.
7. The chimeric receptor of claim 1 wherein the domain further comprises a hinge region.
8. The chimeric receptor of claim 1, wherein the transmembrane domain comprises a CD28 transmembrane domain.
9. The chimeric receptor of claim 1, wherein the intracellular domain comprises a CD28 intracellular domain.
10. The chimeric receptor of claim 1, wherein the intracellular domain comprises a CD28-CD3? intracellular domain.
11. The chimeric receptor of claim 1, wherein the chimeric receptor comprises CD3?.
12. The chimeric receptor of claim 1, wherein the chimeric receptor binds to a molecule comprising Fc.
13. The chimeric receptor of claim 12, wherein the molecule comprising Fc is an IgG antibody.
14. The chimeric receptor of claim 13, wherein the IgG antibody is IgG1 or IgG3.
15. A nucleic acid comprising a nucleotide sequence encoding a chimeric receptor of any one of claims 1-14.
16. The nucleic acid of claim 15, wherein the nucleic acid is an RNA or a cDNA molecule.
17. A vector comprising the nucleic acid of claim 15 or claim 16.
18. The vector of claim 17, wherein the vector is an expression vector.
19. The vector of claim 18, wherein the vector is a viral vector.
20. The vector of claim 19, wherein the viral vector is a retroviral vector.
21. A cell, comprising a nucleic acid of claim 15 or claim 16, or a vector of any one of claims 17-20.
22. The cell of claim 21, wherein the cell is an immune cell.
23. The cell of claim 22, wherein the immune cell is a T cell.
24. The cell of any one of claims 21-23, wherein the cell is a T cell.
25. A pharmaceutical composition, comprising (a) a nucleic acid of claim 15 or claim 16, a vector of any one of claims 17-20, or a cell of any one of claims 21-24, and (b) a pharmaceutically acceptable carrier.
26. The pharmaceutical composition of claim 25, wherein the composition further comprises an Fc-containing therapeutic agent.
27. The pharmaceutical composition of claim 26, wherein the Fc-containing therapeutic agent is an Fc fusion protein or an antibody.
28. The pharmaceutical composition of claim 27, wherein the antibody binds to a cell surface antigen.
29. The pharmaceutical composition of claim 27 or 28, wherein the antibody binds epidermal growth factor receptor.
30. The pharmaceutical composition of claim 29, wherein the antibody is Cetuximab.
31. The pharmaceutical composition of claim 27 or 28, wherein the antibody binds B7-H3.
32. The pharmaceutical composition of claim 31, wherein the antibody is anti-B7-H3 376.96 mAb.
33. The pharmaceutical composition of claim 31, wherein the antibody is Enoblituzumab.
34. A kit, comprising: a first pharmaceutical composition that comprises (i) a nucleic acid of claim 15 or claim 16, a vector of any one of claims 17-20, or a cell of any one of claims 21-24, and (ii) a pharmaceutically acceptable carrier.
35. The kit of claim 34, further comprising a second pharmaceutical composition that comprises an Fc-containing therapeutic agent and a pharmaceutically acceptable carrier.
36. The kit of claim 35, wherein the Fc-containing therapeutic agent is an Fc fusion protein or an antibody.
37. The kit of claim 36, wherein the antibody binds to a cell surface antigen.
38. The kit of claim 36 or 37, wherein the antibody binds epidermal growth factor receptor.
39. The kit of claim 38, wherein the antibody is Cetuximab.
40. The kit of claim 36 or 37, wherein the antibody binds B7-H3.
41. The kit of claim 40, wherein the antibody is anti-B7-H3 376.96 mAb.
42. The kit of claim 40, wherein the antibody is Enoblituzumab.
43. A pharmaceutical composition for use in killing tumor cells or for treating cancer in a subject, the pharmaceutical composition comprising an effective amount of immune cells that express a chimeric receptor of any one of claims 1-14 and a pharmaceutically acceptable carrier.
44. A pharmaceutical composition for use in enhancing antibody-dependent cell-mediated cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP) or for enhancing efficacy of an antibody-based immunotherapy in a subject, the pharmaceutical composition comprising an effective amount of immune cells that express a chimeric receptor of any one of claims 1-14 and a pharmaceutically acceptable carrier.
45. The pharmaceutical composition for use of claim 43 or 44, wherein the immune cells are natural killer cells, macrophages, neutrophils, eosinophils, T cells, or a combination thereof.
46. The pharmaceutical composition for use of claim 45, wherein the immune cell is a T cell.
47. The pharmaceutical composition for use of claim 46, wherein the host immune cells are autologous.
48. The pharmaceutical composition for use of claim 46, wherein the host immune cells are allogeneic.
49. The pharmaceutical composition for use of any one of claims 43-48, wherein the immune cells are activated, expanded, or both ex vivo.
50. The pharmaceutical composition for use of any one of claims 43-49, wherein the subject has been treated or is being treating with an Fc-containing therapeutic agent.
51. The pharmaceutical composition for use of claim 50, wherein the Fc-containing therapeutic agent is a therapeutic antibody or a Fc fusion protein.
52. The pharmaceutical composition for use of claim 51, wherein the Fc-containing therapeutic agent is an antibody.
53. The pharmaceutical composition for use of claim 52, wherein the antibody binds to a cell surface antigen.
54. The pharmaceutical composition for use of claim 52 or 53, wherein the antibody binds epidermal growth factor receptor.
55. The pharmaceutical composition for use of claim 54, wherein the antibody is Cetuximab.
56. The pharmaceutical composition for use of claim 52 or 53, wherein the antibody binds B7-H3.
57. The pharmaceutical composition for use of claim 56, wherein the antibody is anti-B7-H3 376.96 mAb.
58. The pharmaceutical composition for use of claim 56, wherein the antibody is Enoblituzumab.
59. The pharmaceutical composition for use of any one of claims 50-58, wherein the subject is a human patient suffering from a cancer and the Fc-containing therapeutic agent is for treating the cancer.
60. The pharmaceutical composition for use of claim 59, wherein the cancer includes cells expressing epidermal growth factor.
61. The pharmaceutical composition for use of claim 59, wherein the cancer includes cells expressing B7-H3.
62. The pharmaceutical composition for use of claim 59, wherein the cancer is a solid cancer or a hematopoietic cancer.
63. The pharmaceutical composition for use of claim 59, wherein the cancer is a carcinoma, lymphoma, a sarcoma, blastoma, a leukemia, or a cancer stem cell.
64. The pharmaceutical composition for use of claims 60-63, wherein the cancer is acute myeloid leukemia, breast cancer, lung cancer, non small cell lung carcinoma, gastrointestinal cancer, colorectal cancer, head and neck cancer, and glioblastoma multiforme, and glioblastoma multiforme cancer stem cells.
65. The pharmaceutical composition for use of any one of claims 43 and 59-64, wherein the subject's cancer has been irradiated or is being irradiated.
66. The pharmaceutical composition for use of any one of claims 43-65, wherein the subject has one or more tumors and the immune cells migrate to the one or more tumors.
67. A method for preparing immune cells expressing a chimeric receptor, comprising: (i) providing a population of immune cells; (ii) introducing into the immune cells a nucleic acid encoding a chimeric receptor of any one of claims 1-14; and (iii) culturing the immune cells under conditions allowing for expression of the chimeric receptor.
68. The method of claim 67, wherein the population of immune cells are derived from peripheral blood mononuclear cells (PBMC).
69. The method of claim 67 or claim 68, wherein the immune cells are natural killer cells, macrophages, neutrophils, eosinophils, T cells, or a combination thereof.
70. The method of any one of claims 67-69, wherein the immune cells are derived from a human patient.
71. The method of claim 70, wherein the human patient is a cancer patient.
72. The method of any one of claims 67-71, wherein the nucleic acid is a viral vector.
73. The method of claim 72, wherein the viral vector is a retroviral vector.
74. The method of any one of claims 67-71, wherein the nucleic acid is an RNA molecule.
75. The method of any of claims 67-74, wherein the vector is introduced into the immune cells by retroviral transduction or electroporation.
76. A method of treating cancer in a subject, comprising administering to the subject an effective amount of the nucleic acid molecule of any of claims 15-16, the vector of any of claims 17-20, the cell of any of claims 21-24 and/or a pharmaceutical composition of any of claims 25-33, thereby treating cancer in the subject.
77. A method of enhancing ADCC or ADCP or for enhancing efficacy of an antibody-based immunotherapy in a subject having a cancer, comprising administering to the subject an effective amount of the nucleic acid molecule of any of claims 15-16, the vector of any of claims 17-20, the cell of any of claims 21-24 and/or a pharmaceutical composition of any of claims 25-33, thereby enhancing ADCC or ADCP or enhancing efficacy of the antibody-based immunotherapy in the subject.
78. The method of claim 76 or 77, wherein the subject has been treated or is being treating with an Fc-containing therapeutic agent.
79. The method of claim 78, wherein the Fc-containing therapeutic agent is a therapeutic antibody or a Fc fusion protein.
80. The method of claim 79, wherein the Fc-containing therapeutic agent is an antibody.
81. The method of claim 80, wherein the antibody binds to a cell surface antigen.
82. The method of claim 80 or 81, wherein the antibody binds epidermal growth factor receptor.
83. The method of claim 82, wherein the antibody is Cetuximab.
84. The method of claim 80 or 81, wherein the antibody binds B7-H3.
85. The method of claim 84, wherein the antibody is anti-B7-H3 376.96 mAb.
86. The method of claim 84, wherein the antibody is Enoblituzumab.
87. The method of any one of claims 78-86, wherein the Fc-containing therapeutic agent is for treating the cancer.
88. The method any one of claims 76-87, wherein the cancer includes cells expressing epidermal growth factor.
89. The method any one of claims 76-88, wherein the cancer includes cells expressing B7-H3.
90. The method any one of claims 76-89, wherein the cancer is a solid cancer or a hematopoietic cancer.
91. The method any one of claims 76-90, wherein the cancer is a carcinoma, lymphoma, a sarcoma, blastoma, a leukemia, or a cancer stem cell.
92. The method any one of claims 76-91, wherein the subject's cancer has been or is being irradiated.
93. The method any one of claims 76-92, wherein the subject has one or more tumors and the immune cells migrate to the one or more tumors.
94. A chimeric receptor comprising a.) an extracellular ligand binding domain of CD64, wherein the extracellular ligand binding domain of CD64 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:3; b.) a transmembrane domain; and c.) an intracellular domain.
95. A chimeric receptor comprising a.) an extracellular ligand binding domain of CD64, wherein the extracellular ligand binding domain of CD64 comprises one or more of the following: (i) a CD64 D2 domain C-strand (Tyr-133-Leu-136); (ii) a CD64 C-strand (Lys-142-His-148); and/or (iii) a CD64 CE loop (His-148-Trp-149); b.) a transmembrane domain; and c.) an intracellular domain.
96. The chimeric receptor of claim 95, wherein the CD64 D2 domain C-strand comprises amino acid residues Tyr-133 to Leu-136 of CD64 (YNVL).
97. The chimeric receptor of claim 95 or 96, wherein the CD64 C-strand comprises amino acid residues Lys-142 to His-148 of CD64 (KAFKFFH).
98. The chimeric receptor of any one of claims 95-97, wherein the CD64 CE loop comprises amino acid residues His-148 and Trp-149 of CD64 (HW).
99. The chimeric receptor of any one of claims 95-98, wherein the extracellular ligand binding domain of CD64 comprises (i) and (ii), (ii) and (iii), (i) and (iii), or (i), (ii), and (iii).
100. The chimeric receptor of any one of claims 96-99, wherein the numbering of the amino acid residues is relative to SEQ ID NO:21.
101. The chimeric receptor of any one of claims 95-100, wherein the extracellular ligand binding domain of CD64 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:3.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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[0110] A.) Representative bioluminescence image (left panel) of firefly luciferase expressing HCT-116 Luc+ co-cultured with CD64-CR or non-transduced T effector cells at different E:T ratios. Each experimental condition was represented in triplicates. After a 72-hour co-culture, D-luciferin (150 ?g/mL) was added to cell culture medium. Photons (total flux) emitted from HCT-116 Luc+ in the selected regions of interest (ROI) were quantified using the Living Image? software (right panel). ****p<0.0001, two-way ANOVA with Bonferroni test adjusted p value was used for statistical analysis. B.) Representative MTT assay showing viable HCT-116 and HT-29 tumor cells after a 72-hour co-culture with CD64-CR T or non-transduced (NT) T cells at different E:T ratios. Each condition was performed in triplicate and means?SD of optical density measured at 570 nM (Ab 570 nM) are shown. C.) Flow-cytometry plots of HCT-116 and HT-29 CRC cell lines after a 4-hour incubation with CD64-CR T cells at an E:T ratio of 2:1. Non-transduced (NT) T cells were used as a control. Following co-culture, the cells were collected and stained with APC-conjugated anti-human CD3 mAb, FITC-conjugated annexin V and PI. HCT-116 and HT-29 target cells were distinguished from T effector cell by posting an electronic gate on CD3? cells. Percentages of cells were indicated in the quadrants. D.) HCT-116 and HT-29 cell viability tested with trypan-blue exclusion cell count. CRC target cells were co-cultured with CD64-CR T or non-transduced (NT) T cells at the indicated E:T ratio. After an overnight-incubation at 37? C., non-adherent effector T cells were removed and the remaining adherent HCT-116 or HT-29 were counted. Error bars denote SD. Unpaired two-tailed T test was used; *p<0.05, **p<0.01.
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[0112] A.) Determination of residual BJ and HCT-116 cell number following an overnight-incubation with CD64-CR T cells at E:T ratios of 2:1 and 1:1. Non transduced (NT) T cells were used as a control. Error bars indicate SD. Unpaired two-tailed T test was applied; *p<0.05, **p<0.01. B.) Representative contour plots showing viability of human BJ normal fibroblasts (upper panel) or CRC HCT-116 cell line (lower panel) after a 4-hour co-culture of with or without CD64-CR T cells at an E:T ratio of 2:1. The CD3? target cell viability was assessed by annexin V and PI staining and following flow cytometry analysis. Numbers in the quadrants indicate percentage of cells. C.) Non-tumor BJ, IMR-90 fibroblasts, human myoblasts and CRC HCT-116 cells were co-cultured with CD64-CR T and non-transduced (NT) T cells at indicated E:T ratio for 72 hours. The viability of target cells was determined by MTT assay. Each point in the curves indicate mean?SD of absorbance detected at 570 nM (Ab.sub.570) in four independent experiments (N=4) for BJ fibroblasts and HCT116 and two independent experiments (N=2) for IMR-90 and myoblasts. *p<0.05; **p<0.01; ****p<0.0001; two-way-ANOVA with Bonferroni test was used.
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[0114] A.) HCT-116, HT-29 and CaCo-2 CRC cell lines were incubated with CD64-CR T or non-transduced (NT) T cells with or without cetuximab, panitumumab or 396.96 mAb (1 ?g/ml) at different E:T ratio. Following a 72-hour co-culture, the tumor cell viability was assessed by MTT test. Three experimental replicates for each point was performed. Means and SD of the best of three independent donors (N=3) were showed. Ab.sub.570: absorbance at 570 nM. B.) HCT-116 cells were co-cultured with or without CD64-CR T or non-transduced (NT) T cells at an E:T ratio of 2:1 for 4 hours at 37? C. in the presence or absence of 1 ?g/ml of anti-EGFR mAb cetuximab or anti-B7-H3 mAb 376.96. Then, cells were collected and stained with APC-conjugated anti-human CD3, FITC-conjugated annexin V and PI and analyzed by flow cytometry. The numbers in the quadrants indicate percentage of cells.
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[0116] A.) CD64-CR T cells were incubated for 4 hours with or without HCT116 CRC cells at an E:T ratio of 2:1 in the presence or absence of 1 ?g/ml of cetuximab (C), panitumumab (P) or 376.96 mAb. FITC-conjugated mouse anti-human CD107A mAb and 2 ?M of monensin were included during coculture. Then, cells were stained with PE-conjugated mouse anti-human CD64 and analyzed by flow cytometry. MFI=mean fluorescence intensity. Numbers in the quadrants indicate percentages of cells. B.) Colocalization and polarization of perforin on CD64-CR T cells upon conjugation with EGFR positive HCT-116 CRC cells. The indirect immunofluorescence method utilized for these type of experiments is detailed in the methods section. HCT-116 cells were incubated in the presence of CD64-CR T cells at an T:E ratio of 1:2, for 30 minutes at 37? C., formalin fixed, permeabilized, and incubated, overnight at 4? C., in the presence of a rabbit anti-human EGFR mAb or a mouse anti-human CD64 ab. The cell mix was incubated with a Cy-5-conjugated donkey anti-rabbit or a Cy3-conjugated donkey anti-mouse. Then, the cell mix was incubated, for 2 hours at 37? C., with a FITC-conjugated mouse anti-human perforin mAb. The cell mix nuclei were stained by DAPI. The Cells were analyzed as indicated by confocal microscopy.
[0117]
[0118] A.) CD64-CR T cells exerted a superior anti-tumor activity than CD16.sup.158V-CR T cells against HCT-116 cells. HCT-116 CRC cells were cocultured, in triplicate, with CD64-CR or CD16.sup.158V-CR transduced T cells in the presence or absence of 1 ?g/ml of cetuximab or 376.96 mAb at increasing E:T ratios. HCT-116 cell survival was analyzed by MTT assay after 72-hour incubation. Non transduced (NT) T cells were used as a control. Mean?SD are shown. *p<0.05, ****p<0.0001, two-way-ANOVA with Bonferroni test was applied. Ab.sub.570: absorbance at 570 nM. ns: not significant. B.) B7-H3.CAR expression on human T lymphocytes. Activated CD3.sup.+ T cells were transduced with retroviral particle carrying the B7-H3.CAR vector. After a 3-day incubation, the expression of B7-H3.CAR was determined by flow cytometry; dotted line light gray histogram shows control non-transduced (NT) T cells, full line gray histogram indicates B7-H3.CAR transduced T cells. C.) Equivalent activity of CD64-CR T cells in combination with anti-B7-H3 mAb 376.96 and the classic B7-H3.CAR T cells against CRC cell lines. Triplicates of CD64-CR T cells with or without 1 ?g/ml of 376.96 mAb or B7-H3.CAR T cells, obtained from the same donor and cultured in the same conditions, were incubated with HCT-116 or HT-29 target cells at different E:T ratios. Non-transduced (NT) T cells were used as a control. Following a 72-hour incubation at 37? C., MTT assay was performed to determine the viability of the HCT-116 and HT-29 remaining cells. The quantification of absorbance at 570 nM (Ab.sub.570) was showed as mean?SD. ****p<0.0001, two-way-ANOVA with Bonferroni test was used.
[0119]
[0120] A.) Representative flow-cytometry plots showing cetuximab, panitumumab and 376.96 mAb binding on CD64-CR T cells. CD64-CR T cells were incubated with 10 ?g/ml of cetuximab, panitumumab or 376.96 mAb for 30 min at 4? C. in the presence or absence of FcR blocking reagent (BR). The binding of the indicated mAbs on T cell surface was revealed by staining cells with FITC-conjugated mouse anti-human IgG (MAH) (for cetuximab and panitumumab) or FITC-conjugated goat anti-mouse IgG (GAM) (for 376.96) for 30 min at 4? C. Numbers in the quadrants indicate percentage of cells. B.) Dose-response of the cetuximab, panitumumab and 376.96 mAb binding on CD64-CR T cells. CD64-CR transduced T cells were incubated with scaling amounts of cetuximab, panitumumab or 376.96 mAb. Following a 30 min-incubation at 4? C., cells were washed and stained with FITC-conjugated mouse anti-human IgG (MAH) or FITC-conjugated goat anti-mouse IgG (GAM) and analyzed by flow cytometry. The percentage of positive cells (left panel) and mean fluorescence intensity (MFI) (right panel) are shown. C.) Binding of the 376.96 mAb to CD64-CR T cells in the presence or absence of increasing concentrations of heat-inactivated human plasma at 37? C. and 4? C. CD64-CR T cells were incubated with 10 ?g/ml of the 376.96 mAb at the mentioned temperatures. After a 30 min-incubation, T cells were stained with FITC-conjugated anti-mouse IgG at the same temperatures. The percentage of positive cells and the MFI were determined by flow cytometry. D.) The anti-CRC efficacy of the CD64-CR T cells was not affected by the presence of a third-party CD14+ monocytes. Seven days post transduction, CD64-CR T cells were incubated, in triplicate at 37? C., with HCT-116 cells and the indicated mAbs, in the presence or absence of a third party Fc?R+CD14+ monocytes (M) at an effector:target:monocyte (E:T:M) ratio of 1:1:1. Following a 72-hour co-culture, non-adherent T cells were removed and the target cell viability was determined by MTT assay. The absorbance measured at 570 nm (Ab.sub.570) is shown as mean?SD. *p<0.01, ****p<0.0001 were analyzed using two-way-ANOVA with Bonferroni test. NT: non transduced. See, also
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[0130] A.) shows a schematic diagram showing the experiment described in Example 10. Low fluo indicates low fluorescence diet. B.) shows a series of images showing bioluminescent imaging (BLI) and fluorescence imaging (FLI) overlays for the indicated treatment groups for the experiment described in Example 10. These data show that both non-transduced (NT) T cells and CD64-CR T cells labeled with near-infrared cell tracker (NIR) migrate to the tumor site. C.) is a graph showing the BLI signal of cancer cells on day 2 of the experiment described in Example 10.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0131] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.
[0132] The articles a and an are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, an element means one element or more than one element.
[0133] Activation, as used herein, refers to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production, and detectable effector functions. The term activated T cells refers to, among other things, T cells that are undergoing cell division.
[0134] As used herein, the term Chimeric Receptor or alternatively a CR refers to a recombinant polypeptide construct including at least an extracellular binding domain, a transmembrane domain and a cytoplasmic signaling domain including a functional signaling domain derived from a stimulatory molecule as defined below. In one aspect, the stimulatory molecule is the zeta chain associated with the T cell receptor complex.
[0135] As used herein, a signaling domain is the functional portion of a protein which acts by transmitting information within the cell to regulate cellular activity via defined signaling pathways by generating second messengers or functioning as effectors by responding to such messengers.
[0136] The term anti-tumor effect as used herein, refers to a biological effect which can be manifested by various means, including but not limited to, a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, a decrease in tumor cell proliferation, a decrease in tumor cell survival, or amelioration of various physiological symptoms associated with the cancerous condition. An anti-tumor effect can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the invention in prevention of the occurrence of tumor in the first place.
[0137] As used herein, the term autologous is meant to refer to any material derived from the same individual to whom it is later to be re-introduced into the individual.
[0138] Allogeneic refers to any material derived from a different animal of the same species as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically.
[0139] The term cancer as used herein is defined as disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like, including other cancers described herein.
[0140] As used herein, the term conservative sequence modifications is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody or antibody fragment of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CD64 chimeric receptor can be replaced with other amino acid residues from the same side chain family and the altered receptor can be tested for the ability to bind, for example, Fc, using the standard functional assays.
[0141] As used herein, the term percent (%) sequence identity, with respect to a reference polynucleotide or polypeptide sequence, is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, percent sequence identity values may be generated using the sequence comparison computer program BLAST. As an illustration, the percent sequence identity of a given nucleic acid or amino acid sequence, A, to, with, or against a given nucleic acid or amino acid sequence, B, (which can alternatively be phrased as a given nucleic acid or amino acid sequence, A that has a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence, B) is calculated as follows:
100 multiplied by (the fraction X/Y)
[0142] where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program's alignment of A and B, and where Y is the total number of nucleic acids in B. It will be appreciated that where the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A.
[0143] A disease is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate. In contrast, a disorder in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health
[0144] By the term stimulation, is meant a primary response induced by binding of a stimulatory molecule (e.g., a TCR/CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex. Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-?, and/or reorganization of cytoskeletal structures, and the like.
[0145] A stimulatory molecule, means a molecule expressed by a T cell that provide the primary cytoplasmic signaling sequence(s) that regulate primary activation of the TCR complex in a stimulatory way for at least some aspect of the T cell signaling pathway. In one aspect, the primary signal is initiated by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, and which leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM containing primary cytoplasmic signaling sequences that are of particular use in the invention include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as ICOS) and CD66d. In a specific CR of the invention, the cytoplasmic signaling molecule in any one or more CRs of the invention, including CRs includes a cytoplasmic signaling sequence derived from CD3-zeta.
[0146] A costimulatory molecule refers to the cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient immune response. Costimulatory molecules include, but are not limited to an MHC class I molecule, BTLA and a Toll ligand receptor, as well as OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD1 Ia/CD 18) and 4-1BB (CD137).
[0147] An effective amount as used herein, means an amount which provides a therapeutic or prophylactic benefit.
[0148] Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i. e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
[0149] Unless otherwise specified, a nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or a RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).
[0150] As used herein endogenous refers to any material from or produced inside an organism, cell, tissue or system.
[0151] As used herein, the term exogenous refers to any material introduced from or produced outside an organism, cell, tissue or system.
[0152] The term expression as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.
[0153] The term nucleic acid or polynucleotide refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine.
[0154] The terms patient, subject, individual, and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.
[0155] As used herein, the terms peptide, polypeptide, and protein are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can include a protein's or peptide's sequence. Polypeptides include any peptide or protein including two or more amino acids joined to each other by peptide bonds.
[0156] As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
[0157] By the term specifically binds, as used herein with respect to an antibody, is meant an antibody or antibody fragment which recognizes and binds with a specific antigen, but does not substantially recognize or bind other molecules in a sample.
[0158] The term therapeutic as used herein means a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.
[0159] The term therapeutically effective amount refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term therapeutically effective amount includes that amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.
[0160] To treat a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.
[0161] The term transfected or transformed or transduced as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into a host cell. A transfected or transformed or transduced cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.
[0162] A vector is a composition of matter which includes an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term vector includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.
[0163] As is discussed below, we have designed a CD64-chimeric receptor (CD64-CR) composed of the human Fc?RI extracellular binding domain linked to an intracellular tail capable of inducing a cytotoxic T cell response upon stimulation. The CD64-recombinant receptor was expressed on human T cells and its anti-tumor activity was tested, in vitro and in vivo, against colorectal carcinoma (CRC) cell lines in the presence or absence of anti-epidermal growth factor receptor (EGFR) mAbs or anti-B7-H3 mAb (376.96). The CD64-CR engineered T cells were efficiently redirected against CRC tumor cells and an MHC-unrestricted target cell killing was observed regardless of antibody ligation.
[0164] Fc?-chimeric receptors (CRs), in particular, have been designed to redirect T cells against cancer cells by monoclonal antibodies (mAbs) with specificity for cell surface tumor-associated-antigens. Since Fc?Rs bind the Fc fragment of immunoglobulins G (IgGs), the antibody-dependent-cellular-cytotoxicicty (ADCC) is the expected mechanism by which Fc?-CR T cells kill cancer cells. However, recent evidence suggests that, other then the Fc fragment of IgG, Fc?Rs bind also pentraxins. The aim of this study is to investigate whether Fc?? (CD64) T cells act as biosensors capable of detecting unknown Fc?R ligand(s) on the cell surface of cancer cells. Healthy, peripheral blood (PB) T cells were transduced with a retroviral vector coding for a human CD64-CR in which the human leader sequence (LS) was replaced with a mouse LS (mLS-CD64/CD28/z) since the latter was the best construct in inducing a satisfactory level of T cell trasduction. The engineered T cells rapidly expanded, in vitro, reaching an average of T cell transduction up to 60% on the third week of culture. CD64-CR,CD8 T cells mainly showed a central memory phenotype. Importantly, CD64-CR T cells sensed either B7-H3 positive colorectal carcinoma cell lines (HCT-116 and HT-29) or head and neck carcinoma cells (FaDu and A253) or a variety of additional cell lines (shown in Table 3). Following conjugation with cancer cells, CD64-CR triggered a powerful HLA-unrestricetd T cell-mediated cytotoxicity leading to a strong cancer cell elimination, in vitro, even at very low level of E:T cell ratio. These data evidenced that CD64-CR recognized unknown cell surface ligand(s) on cancer cells. CD64-CR T cells preferentially recognized cancer cells since marginally affected the viability of skin fibroblasts and myoblast and to a lesser extent lung fibroblasts, particularly when utilized at low E:T ratio. Confocal microscopy studies strongly suggested that CD64-CR T cells, used at least the CD64-CR T cell killing machiney to exert their cytotoxic effects. This is supported by laboratory data in which CD64-CR polarized and cololocalized with perforine, in the presence of cancer cells. Furthermore, the oponization of the cell surface of the cancer cells with an anti-B7-H3, 376.96 or cetuximab mAb resulted in a strong enhancement of the CD64-CR T cell-mediated cytotoxicity. Thus, CD64-CR confers T cells with a NK like cytotoxic function involving both an endogenous antibody independent cytotoxicity and ADCC which was triggered by the anti-B7-H3 mAb. The cytotoxic potential of CD64-CR T was evaluated by a direct comparison with that of the CD16-CR or a conventional B7-H3-CAR. CD64-CR T cells and B7-H3-CAR T cells mediated an equivalent extent of cytotoxicity on all cancer cells utilized. In contrast, CD16-CR T cells killed only in the presence of cetuximab. Overall, CD64-CR T cells mediate an NK like activity consisting of an HLA-unrestricted cellular cytotoxicity, which is optimized by the use of the 376.96 mAb. These cells preferentially killed cancer cells. As a result, such a combination could be used to develop a rational cell-based immunotherapy of B7-H3 positive and negative solid and hematopoietic cancer cells.
Chimeric Receptors (CRs)
[0165] In one embodiment, the present invention relates to a chimeric receptor (CR). The CR includes an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding element otherwise referred to as a binding domain. In certain embodiments, the intracellular domain or otherwise the cytoplasmic domain includes, a costimulatory signaling region and a zeta chain portion. The costimulatory signaling region refers to a portion of the CR including the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient response of lymphocytes.
[0166] Between the extracellular domain and the transmembrane domain of the CR, or between the cytoplasmic domain and the transmembrane domain of the CR, there may be optionally incorporated a spacer domain. As used herein, the term spacer domain generally means any oligo- or polypeptide that functions to link the transmembrane domain to, either the extracellular domain or, the cytoplasmic domain in the polypeptide chain. A spacer domain may include up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids.
[0167] The extracellular domain, transmembrane domain, and intracellular domain can be derived from any desired source of such domains.
[0168] In one embodiment, the CR includes an amino acid sequence as follows:
TABLE-US-00001 (MS-SP)-CD64.28.? (SEQIDNO:1) MGILTSFGDDMWLLTTLLLWVPVGGQVDTTKAVITLQPPWVSVFQEETVT LHCEVLHLPGSSSTQWFLNGTATQTSTPSYRITSASVNDSGEYRCQRGLS GRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWKDKLVYNVLYYRN GKAFKFFHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISVTVKELFP APVLNASVTSPLLEGNLVTLSCETKLLLQRPGLQLYFSFYMGSKTLRGRN TSSEYQILTARREDSGLYWCEAATEDGNVLKRSPELELQVLGLQLPTPVW FHPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNM TPRRPGPTRKHYQPYAPPRDFAAYRSVDRVKFSRSADAPAYQQGQNQLYN ELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
[0169] In another embodiment, the CR includes an amino acid sequence as follows:
TABLE-US-00002 CD64.28.? (SEQIDNO:8) MGFLTTLLLWVPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPG SSSTQWFLNGTATQTSTPSYRITSASVNDSGEYRCQRGLSGRSDPIQLEI HRGWLLLQVSSRVFTEGEPLALRCHAWKDKLVYNVLYYRNGKAFKFFHWN SNLTILKTNISHNGTYHCSGMGKHRYTSAGISVTVKELFPAPVLNASVTS PLLEGNLVTLSCETKLLLQRPGLQLYFSFYMGSKTLRGRNTSSEYQILTA RREDSGLYWCEAATEDGNVLKRSPELELQVLGLQLPTPVWFHPGPSKPFW VLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRK HYQPYAPPRDFAAYRSVDRVKFSRSADAPAYQQGQNQLYNELNLGRREEY DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR GKGHDGLYQGLSTATKDTYDALHMQALPPR*
[0170] In yet another embodiment, the CR includes an amino acid sequence as follow:
TABLE-US-00003 CD64(EC-TM).28.? (SEQIDNO:15) MGFLTTLLLWVPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPG SSSTQWFLNGTATQTSTPSYRITSASVNDSGEYRCQRGLSGRSDPIQLEI HRGWLLLQVSSRVFTEGEPLALRCHAWKDKLVYNVLYYRNGKAFKFFHWN SNLTILKTNISHNGTYHCSGMGKHRYTSAGISVTVKELFPAPVLNASVTS PLLEGNLVTLSCETKLLLQRPGLQLYFSFYMGSKTLRGRNTSSEYQILTA RREDSGLYWCEAATEDGNVLKRSPELELQVLGLQLPTPVWFHVLFYLAVG IMFLVNTVLWVTIVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDF AAYRSVDRVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGL STATKDTYDALHMQALPPR
Leader Sequence
[0171] In one embodiment, the CR includes a leader sequence. Exemplary leader sequences include a mouse CD64 or human CD64 leader sequences.
[0172] The mouse CD64 leader sequence, for example, includes the following amino acids:
TABLE-US-00004 (SEQIDNO:2) MGILTSFGDDMWLLTTLLLWVPVGG
[0173] The CD64 human leader sequence, for example, includes the following amino a
TABLE-US-00005 (SEQIDNO:9) MGFLTTLLLWVPVDG
Binding Domain
[0174] The binding domain may be obtained from any of the wide variety of extracellular domains or secreted proteins associated with ligand binding and/or signal transduction.
[0175] The present invention, in one embodiment, includes a binding domain of CD64. CD64 is a type of integral membrane glycoprotein known as an Fc receptor that binds monomeric IgG-type antibodies with high affinity. It is also known as Fc-gamma receptor (Fc?RI). After binding IgG, CD64 interacts with an accessory chain known as the common ? chain (? chain), which possesses an ITAM motif that is necessary for triggering cellular activation.
[0176] Structurally CD64 is composed of a signal peptide that allows its transport to the surface of a cell, three extracellular immunoglobulin domains of the C2-type that it uses to bind antibody, a hydrophobic transmembrane domain, and a short cytoplasmic tail. CD64 is the Fc?R with high-affinity for monomeric IgGs and, unlike other members, it is composed of three extracellular Ig-like domains (D1, D2, and D3). CD64 is expressed on monocytes/macrophages and dendritic cells (DCs) while neutrophils and mast cells express CD64 after stimulation with interferon-? (IFN?) or granulocyte colony-stimulating factor (G-CSF). The engagement of CD64 on effector cells results in ADCC and antibody-dependent cellular phagocytosis (ADCP) of target cells.
[0177] The present invention includes a binding domain that binds that binds monomeric IgG-type antibodies with high affinity. The CR of the invention can be engineered to include any CD moiety that is specific to binding antibodies. The binding domain can be any domain that binds to any antibody including including but not limited to monomeric IgG-type antibodies, monoclonal antibodies, polyclonal antibodies, synthetic antibodies, scFvs, human antibodies, humanized antibodies, and fragments thereof.
[0178] In one embodiment, the CD64 binding domain includes the following amino acids:
TABLE-US-00006 (SEQIDNO:3) QVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTATQT STPSYRITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVFT EGEPLALRCHAWKDKLVYNVLYYRNGKAFKFFHWNSNLTILKTNISHNGT YHCSGMGKHRYTSAGISVTVKELFPAPVLNASVTSPLLEGNLVTLSCETK LLLQRPGLQLYFSFYMGSKTLRGRNTSSEYQILTARREDSGLYWCEAATE DGNVLKRSPELELQVLGLQLPTPVWFH
[0179] Other binding domain regions useful for engineering CD64 CRs include those found at the interface of Fc?RI with the lower hinge Fc region (Lu et al., PNAS 112: 823-838, 2015). The interface between the receptor and the two Fc chains can be divided into three regions: between the Fc?RI D2 domain C-strand (Tyr-133-Leu-136), C-strand (Lys-142-His-148), and CE loop (His-148-Trp-149) and the lower hinge region from the Fc A-chain; between the receptor D1-D2 interdomain hinge (Arg-102-Trp-104), D2 domain BC loop (Trp-127-Tyr-133), and lower hinge from the Fc B-chain; and between the Fc?RI D2 domain FG loop (Met-171-Tyr-176) and the Fc glycans. Accordingly, useful CD64-CR receptors typically include one or more of the Fc?RI D2 domain C-strand (Tyr-133-Leu-136), C-strand (Lys-142-His-148), and CE loop (His-148-Trp-149). In some examples, the numbering of these amino acid sequences is relative to the numbering of the exemplary CD64 amino acid sequence set forth in Uniprot Accession No. P12314-1, reproduced below:
TABLE-US-00007 (SEQIDNO:21) 10 20 30 40 50 MWFLTTLLLW VPVDGQVDTT KAVITLQPPW VSVFQEETVT LHCEVLHLPG 60 70 80 90 100 SSSTQWELNG TATQTSTPSY RITSASVNDS GEYRCQRGLS GRSDPIQLEI 110 120 130 140 150 HRGWLLLQVS SRVFTEGEPL ALRCHAWKDK LVYNVLYYRN GKAFKFFHWN 160 170 180 190 200 SNLTILKTNI SHNGTYHCSG MGKHRYTSAG ISVTVKELFP APVLNASVTS 210 220 230 240 250 PLLEGNLVTL SCETKLLLQR PGLQLYFSFY MGSKTLRGRN TSSEYQILTA 260 270 280 290 300 RREDSGLYWC EAATEDGNVL KRSPELELQV LGLQLPTPVW FHVLFYLAVG 310 320 330 340 350 IMFLVNTVLW VTIRKELKRK KKWDLEISLD SGHEKKVISS LQEDRHLEEE 360 370 LKCQEQKEEQ LQEGVHRKEP QGAT.
Transmembrane Domain
[0180] With respect to the transmembrane domain, the CR can be designed to include a transmembrane domain that is fused to the extracellular domain of the CR. In one embodiment, the transmembrane domain that naturally is associated with one of the domains of CD64 in the CR is used. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
[0181] The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Transmembrane regions of particular use in this invention may be derived from (i.e. include at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD 154. Alternatively, the transmembrane domain may be synthetic, in which case it will include predominantly hydrophobic residues such as leucine and valine. Preferably a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CR. A glycine-serine doublet provides a particularly suitable linker.
[0182] In one embodiment, the transmembrane domain, for example, includes the following amino acids of the CD28 transmembrane domain:
TABLE-US-00008 (SEQIDNO:5) FWVLVVVGGVLACYSLLVTVAFIIFWV
[0183] In one embodiment, the transmembrane domain, for example, includes the CD64 transmembrane domain:
TABLE-US-00009 (SEQIDNO:18) VLFYLAVGIMFLVNTVLWVTI
[0184] In one embodiment, the transmembrane domain, for example, includes the CD64 transmembrane domain with a valine:
TABLE-US-00010 (SEQIDNO:22) VLFYLAVGIMFLVNTVLWVTIV
Intracellular Domain
[0185] The cytoplasmic domain or otherwise the intracellular domain of the CR of the invention is responsible for activation of at least one of the normal effector functions of the immune cell in which the CR has been placed in. The term effector function refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Thus, the term intracellular signaling domain refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular domain is thus meant to include any truncated portion of the intracellular domain sufficient to transduce the effector function signal.
[0186] Preferred examples of intracellular domains for use in the CR of the invention include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.
[0187] It is known that signals generated through the TCR alone are insufficient for full activation of the T cell and that a secondary or co-stimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct classes of intracellular signaling sequence: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences).
[0188] Primary intracellular signaling sequences regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary intracellular signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs.
[0189] Examples of ITAM containing primary intracellular signaling sequences that are of particular use in the invention include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. It is particularly preferred that intracellular signaling molecule in the CR of the invention includes an intracellular signaling sequence derived from CD3 zeta.
[0190] In a preferred embodiment, the intracellular domain of the CR can be designed to include the CD3-zeta signaling domain by itself or combined with any other desired cytoplasmic domain(s) useful in the context of the CR of the invention. For example, the intracellular domain of the CR includes a CD3 zeta chain portion and a costimulatory signaling region. The costimulatory signaling region refers to a portion of the CR including the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4-1 BB (CD137), OX40, CD30, CD40, 4-1 BB, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, and the like. Thus, while the invention in exemplified primarily with CD28 as the co-stimulatory signaling element, other costimulatory elements are within the scope of the invention.
[0191] The intracellular signaling sequences within the intracellular domain of the CR of the invention may be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage. A glycine-serine doublet provides a particularly suitable linker.
[0192] In one embodiment, the intracellular domain is designed to include the signaling domain of CD3-zeta and the signaling domain of CD28.
TABLE-US-00011 CD3-zetasignalingdomain (SEQIDNO:7) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR. CD28signalingdomain Inoneembodiment,theintracellulardomain includetheaminoacidsequence (SEQIDNO:13) RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS
[0193] In other embodiments, the intracellular domain, for example, includes CD28 and CD3-zeta signaling domains.
TABLE-US-00012 (SEQIDNO:23) CD28andCD3-zetasignalingdomains RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSVDRVKFSR SAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR
Sources of Immune Cells
[0194] In certain embodiments, prior to expansion, a source of immune cells (e.g., a T cell or other immune cell described herein) is obtained from a subject. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Preferably, the subject is a human. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, and tumors. In certain embodiments, any number of T cell lines available in the art, may be used. In certain embodiments, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll separation. In one embodiment, cells from the circulating blood of an individual are obtained by apheresis or leukapheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. The cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media, such as phosphate buffered saline (PBS) or wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations, for subsequent processing steps. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.
[0195] In another embodiment, T cells are isolated from peripheral blood by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a
[0196] PERCOLL? gradient. Alternatively, T cells can be isolated from umbilical cord. In any event, a specific subpopulation of T cells can be further isolated by positive or negative selection techniques.
[0197] The cord blood mononuclear cells so isolated can be depleted of cells expressing certain antigens, including, but not limited to, CD34, CD8, CD14, CD19 and CD56.
[0198] Depletion of these cells can be accomplished using an isolated antibody, a biological sample including an antibody, such as ascites, an antibody bound to a physical support, and a cell bound antibody.
[0199] Enrichment of a T cell population by negative selection can be accomplished using a combination of antibodies directed to surface markers unique to the negatively selected cells. A preferred method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4.sup.+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDI Ib, CD16, HLA-DR, and CD8.
[0200] For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells/ml is used. In one embodiment, a concentration of 1 billion cells/nil is used. In a further embodiment, greater than 100 million cells/ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, 01100 million cells/ml is used. In further embodiments, concentrations of 125 or 150 million cells/ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion.
[0201] T cells can also be frozen after the washing step, which does not require the monocyte-removal step. While not wishing to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, in a non-limiting example, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or other suitable cell freezing media. The cells are then frozen to ?80? C. at a rate of 1? per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at ?20? C. or in liquid nitrogen.
[0202] In one embodiment, the population of T cells is comprised within cells such as peripheral blood mononuclear cells, cord blood cells, a purified population of T cells, and a T cell line. In another embodiment, peripheral blood mononuclear cells include the population of T cells. In yet another embodiment, purified T cells include the population of T cells.
Cell Expansion
[0203] In certain embodiments, the immune cells (e.g., a T cell or other immune cell) disclosed herein can be multiplied by about 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600 fold, 700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold, 3000 fold, 4000 fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000 fold, 10,000 fold, 100,000 fold, 1,000,000 fold, 10,000,000 fold, or greater, and any and all whole or partial integers therebetween. In one embodiment, the T cells expand in the range of about 20 fold to about 50 fold.
[0204] Following culturing, the T cells can be incubated in cell medium in a culture apparatus for a period of time or until the cells reach confluency or high cell density for optimal passage before passing the cells to another culture apparatus. The culturing apparatus can be of any culture apparatus commonly used for culturing cells in vitro. Preferably, the level of confluence is 70% or greater before passing the cells to another culture apparatus. More preferably, the level of confluence is 90% or greater. A period of time can be any time suitable for the culture of cells in vitro. The T cell medium may be replaced during the culture of the T cells at any time. Preferably, the T cell medium is replaced about every 2 to 3 days. The T cells are then harvested from the culture apparatus whereupon the T cells can be used immediately or cryopreserved to be stored for use at a later time. In one embodiment, the invention includes cryopreserving the expanded T cells. The cryopreserved T cells are thawed prior to introducing nucleic acids into the T cell.
[0205] In another embodiment, the method includes isolating T cells and expanding the T cells. In another embodiment, the invention further includes cryopreserving the T cells prior to expansion. In yet another embodiment, the cryopreserved T cells are thawed for electroporation with the RNA encoding the chimeric membrane protein.
[0206] Procedures for ex vivo expansion and culture of cells are known in the art.
[0207] Further a medium used to culture the T cells may include an agent that can co-stimulate the T cells. For example, an agent that can stimulate CD3 is an antibody to CD3, and an agent that can stimulate CD28 is an antibody to CD28.
Pharmaceutical Compositions
[0208] Pharmaceutical compositions of the present invention may include the modified immune cell (e.g., a T cell or other immune cell) as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the present invention are preferably formulated for intravenous administration.
[0209] Pharmaceutical compositions of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.
[0210] The cells of the invention to be administered may be autologous, allogeneic or xenogeneic with respect to the subject undergoing therapy.
[0211] Cells of the invention can be administered in dosages and routes and at times to be determined in appropriate pre-clinical and clinical experimentation and trials. Cell compositions may be administered multiple times at dosages within these ranges.
[0212] Administration of the cells of the invention may be combined with other methods useful to treat the desired disease or condition as determined by those of skill in the art.
[0213] It can generally be stated that a pharmaceutical composition including the modified immune cells (e.g., a T cell or other immune cell) described herein may be administered at a dosage of 10.sup.4 to 10.sup.9 cells/kg body weight, in some instances 10.sup.5 to 10.sup.6 cells/kg body weight, including all integer values within those ranges. T cell compositions, for example, may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.
[0214] The administration of the modified T cells, for example, may be carried out in any convenient manner known to those of skill in the art. The cells of the present invention may be administered to a subject by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In other instances, the cells of the invention are injected directly into a site of inflammation in the subject, a local disease site in the subject, a lymph node, an organ, a tumor, and the like.
[0215] It should be understood that the method and compositions that would be useful in the present invention are not limited to the particular formulations set forth in the examples. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the cells, expansion and culture methods, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.
[0216] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.
Therapy
[0217] Host cells (e.g., immune cells) expressing CRs (the encoding nucleic acids or vectors including such) described herein are useful for enhancing ADCC or ADCP in a subject and/or for enhancing the efficacy of an antibody-based immunotherapy. In some embodiments, the subject is a mammal, such as a human. In some embodiments, the subject is a human cancer patient. In some embodiments, the subject has been treated or is being treated with any of the therapeutic antibodies described herein.
[0218] The immune cells are mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition.
[0219] Typically, an effective amount of the immune cells expressing any of the CR constructs described herein are administered into a subject in need of the treatment. The immune cells may be autologous to the subject, i.e., the immune cells are obtained from the subject in need of the treatment, genetically engineered for expression of the CR constructs, and then administered to the same subject. Alternatively, the host cells are allogeneic cells, i.e., the cells are obtained from a first subject, genetically engineered for expression of the CR construct, and administered to a second subject that is different from the first subject but of the same species. For example, allogeneic immune cells may be derived from a human donor and administered to a human recipient who is different from the donor.
[0220] In some embodiments, the immune cells are administered to a subject in an amount effective in enhancing ADCC activity by least 10% or 20%, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or more.
[0221] In some embodiments, the immune cells are co-administered with a therapeutic Fc-containing therapeutic agent (e.g., an antibody or Fc fusion molecule such as Fc fusion protein) to enhance the efficacy of the immunotherapy.
[0222] In any of the methods described herein, the immune cells such as the T lymphocytes are autologous cells isolated from the subject who is subject to the treatment. In one specific embodiment, prior to re-introduction into the subject, the autologous immune cells (e.g., T lymphocytes) are activated and/or expanded ex vivo. In another embodiment, the immune cells (e.g., T lymphocytes or NK cells) are allogeneic cells.
[0223] The CRs of the disclosure may be used for treatment of any cancer, including, without limitation, those for which a specific antibody with an Fc portion that binds to the Fc binder in the CR exists or is capable of being generated.
[0224] Typically, an effective amount of the immune cells expressing CRs, Fc-containing therapeutic agents (e.g., Fc-containing therapeutic proteins such as Fc fusion proteins and therapeutic antibodies), or compositions thereof is administered to a subject (e.g., a human cancer patient) in need of the treatment via a suitable route, such as intravenous administration. Any of the immune cells expressing CRs, Fc-containing therapeutic agents, or compositions thereof may be administered to a subject in an effective amount. As used herein, an effective amount refers to the amount of the respective agent (e.g., the host cells expressing CRs, Fc-containing therapeutic agents, or compositions thereof) that upon administration confers a therapeutic effect on the subject. Determination of whether an amount of the cells or compositions described herein achieved the therapeutic effect is apparent to one of skill in the art.
[0225] In some embodiments, the subject is a human cancer patient. For example, the subject are a human patient suffering from carcinoma, lymphoma, sarcoma, blastoma, or leukemia. Examples of cancers for which administration of the cells and compositions disclosed herein may be suitable include, for example, a wide range of hematologic and solid B7-H3 positive tumors (such as colorectal cancer, breast cancer, non-small cell lung cancer, head and neck cancer, glioblastoma, leukemia, myeloma).
[0226] In accordance with the present disclosure, patients are treated by infusing therapeutically effective doses of immune cells such as T lymphocytes including a CR described. Infusions are repeated as until the desired response is achieved.
[0227] In some embodiments, the immune cells expressing any of the CRs disclosed herein are administered to a subject who has been treated or is being treated with an Fe-containing therapeutic agent (e.g., an Fc-fusion protein or a therapeutic antibody). The immune cells expressing any one of the CRs disclosed herein may be co-administered with an Fe-containing therapeutic agent. For example, the immune cells may be administered to a human subject simultaneously with a therapeutic antibody.
[0228] Alternatively, the immune cells may be administered to a human subject during the course of an antibody-based immunotherapy.
[0229] Examples of therapeutic Fe-containing therapeutic agent include, without limitation, any antibody that binds a cell surface antigen, e.g., cetuximab, enoblituzumab, or other therapeutic antibody used in cancer treatment (for example, rituximab, HERCEPTIN? (trastuzumab), daratumumab, mogamolizumab, panitumumab, and atezolizumab).
[0230] The administration of a Fe-containing therapeutic agent is performed by any suitable route, including systemic administration as well as administration directly to the site of the disease (e.g., to primary tumor).
[0231] The compositions and methods described in the present disclosure may be utilized in conjunction with other types of therapy for cancer, such as chemotherapy, surgery, radiation, gene therapy, and so forth. Such therapies are administered simultaneously or sequentially (in any order) with the immunotherapy according to the present disclosure.
EXAMPLES
Example 1the Efficient Expression of Human CD64-Chimeric Receptor (CR) in T Lymphocytes Required Mouse CD64 Signal Peptide
[0232] Four different CD64 chimeric receptors (CRs) were designed and generated (
[0233] The four CD64 chimeric receptor variants were cloned into retroviral vectors and transduced in human T cells. After transduction, the expression of the chimeric receptors was tested by flow cytometry (
[0234] The distribution of the CD64-CR on the CD4+ and CD8+ T cell subsets was also analyzed. As expected, the CD4/CD8 T cell ratio was inverted during in vitro culture. Four days after transduction, the CD64+CD4+ cell proportion was higher than that of CD64+CD8+ cells (75.6% vs 15.7% respectively) while at 18 days from transduction the percentage of CD64+CD8+ overcame the percentage of CD64+CD4+ cells (82.7% vs 8.48% respectively) (
[0235] The phenotypic analysis, performed by flow cytometry at 30 days after activation, revealed that CD64+ T cells contained 27.4% of stem cell memory (CD45RA+CCR7+CD62L+), 7.02% of central memory (CD45RA?CCR7+CD62L+), 24.2% of effector memory (CD45RA?CCR7?CD62L?), and 11.5% of effector T cells (CD45RA+CCR7?CD62L?) (
Example 2CD64-CR T Cell Mediated the Direct Elimination of CRC Cell Lines Independent of Antibody Ligation
[0236] The high-affinity Fc?RI/CD64 receptor is broadly known for its role in mediating ADCC and ADCP by monocytes/macrophages upon mAb binding. Here, we described an alternative ability of the CD64-CR consisting of a direct tumor target cell recognition and elimination, without the need of an antibody link. HCT-116 Luc+, expressing the firefly luciferase protein, were co-cultured at 37? C. with CD64-CR transduced and control non transduced (NT) T cells at different E:T ratios (
[0237] The cell count experiments showed a significant elimination of HCT-116 and, to a lesser extent, HT29 cells after coculture with CD64-CR T cells, indicating, in line with flow cytometry cytotoxicity data, that CD64-CR T cells killed CRC target cells.
Example 3CD64-CR T Cells Showed Limited Toxic Effect on Human Normal Cells In Vitro
[0238] The direct damage induced on CRC cell lines by CD64-CR T cells suggests the presence of an unknown ligand on sensitive tumor cells. Thus, it is critical to determine whether non-tumor normal cells could be affected by CD64-CR T cells. We tested the potential toxicity of CD64-CR T lymphocytes on normal human skin fibroblast BJ, lung fibroblast IMR-90 cell lines and human primary myoblasts in vitro (
Example 4the Cytotoxic Effect of CD64-CR T Cells Against CRC Cells is Enhanced by the Addition of Cetuximab or 376.96 Anti-B7-H3 mAb
[0239] The ability of CD64-CR T cells to mediate ADCC against CRC cell lines was investigated. The Fc mAb binding assay revealed that CD64-CR specifically bound the Fc fragment of anti-EGFR IgG1 cetuximab and anti-B7-H3 IgG2A 376.96 at 4? C. (
[0240] As known, the CD64 is the only Fc?R that binds monomeric immunoglobulins (Ig) with high affinity. For that reason, we tested the possibility that free serum IgGs could saturate the CD64-CR, thus hampering the binding of therapeutic mAbs. Therefore, the binding of 376.96 mAb to CD64-CR was tested in the presence of increasing concentration of heat-inactivated human plasma both at 4? C. and 37? C. (
[0241] To determine whether the CD64-CR ability of mAb binding could be associated with a functional activity as ADCC, the CD64-transduced T cells were cocultured with EGFR+B7-H3+HCT-116, HT-29 and Caco-2 target cells in the presence of cetuximab, panitumumab or 376.96. After 72-hours incubation, we found that CD64-CR both directly impaired HCT-116 and HT-29 cell viability and exerted ADCC in the presence of cetuximab or 376.96 mAb (
[0242] Furthermore, the direct cytotoxicity and the ADCC elicited by CD64-CR T cells on CRC HCT-116 cell line was also confirmed using a flow-cytometry assay (
Example 5Comparison of CD64-CR with CD16.SUP.158V.-CR and B7-H3.CAR
[0243] In our previous study, we demonstrated the ability of CD16.sup.158V-CR T cells to overcome the resistance to cetuximab of KRAS-mutated CRC HCT-116 and NSCL A549 cell lines (Arriga et al., Int J Cancer 2020; 146:2531-8). Here, we compared the efficiency of T cells containing CD64-CR with the T cells, from the same donor, expressing CD16.sup.158V-CR in HCT-116 cell eliminating, with or without cetuximab or 376.96 mAb. After retroviral transduction, transduced and NT T cells were incubated with target cells for 72 hours at 37? C.
Examples 6 and 7Cytotoxicity
[0244] To define the mechanism underlying the cytotoxic effect exerted by CD64-CR T cells against CRC cells, we investigated the CD107A release by CD64-CR T cells, cultured with target cells, in the presence or absence of mAbs specific for cell surface TAAs. Upon conjugation with HCT-116 cells, CD64-CR T cells mobilized CD107A. Importantly, the CD107A release was enhanced in the presence of cetuximab and the 376.96 mAb. In addition, both cetuximab and 376.96 mAbs induced down-regulation of the CD64-CR. In contrast, panitumumab did not enhance the anti-CD107A mobilization and did not down-regulate the CD64-CR molecule. The 376.96 seemed to be more powerful than cetuximab in term of the extent of CD107A mobilization. Then, we explored the involvement of the CD64-CR T cell lytic machinery by evaluating the distribution of perforin in relationship with CD64-CRs in the CD64-CR T cells. HCT-116 cells were incubated with CD64-CR expressing T cells for 30 min, at 37? C., at an E:T of 2:1, We found that CD64 polarized and colocalized with perforine at the cell surface of the CD64-CR T cells suggesting that a granule-dependent cytotoxicity may be involved (
Example 8Direct CD64-T Cell Cytotoxicity is Antibody-Independent
[0245]
[0246]
[0247]
[0248]
Example 10Results of the Biodistribution of CD64-CR T Cells In Vivo
[0249] To investigate the biodistribution of CD64-CR T cells in vivo, we first injected five hundred thousand FaDu-luciferase positive (Luc+) cells subcutaneously (s.c.) into the dorsal neck of 15 CB17 SCID mice. To avoid excessive autofluorescence, mice were fed with a low fluorescence diet. Following 10 days, mice received intraperitoneal (i.p.) injections of 180 ?l of saline with or without 180 ?g of 376.96 mAb, followed by injection of 2.5?10.sup.6 T cells labeled with near-infrared cell tracker (NIR), 30 min later. On day ?3, endogenous NK cells were neutralized by an i.p. injection of anti-asialo-GM1 antibody. On day 1, imaging analysis was performed and on day 2, mice were sacrificed for an ex vivo study (see
[0250] Animals were divided into 5 groups: group 1: untreated; group 2: non-transduced (NT) T cells-NIR; group 3: CD64-CR T cells-NIR; group 4: NT T cells-NIR+376.96 mAb; and group 5: CD64-CR T cells-NIR+376.96 mAb (see
Materials and Methods
[0251] The above Examples were performed using the following materials and methods.
Antibodies and Reagents
[0252] Allophycocyanin-conjugated mouse anti-human CCR7 Clone 2L1A (566762), fluorescein isothiocyanate-conjugated mouse anti-human CD3 Clone UCHT1 (555332), PerCP-Cy?5.5-conjugated mouse anti-human CD4 Clone SK3 (332772), APC-conjugated mouse anti-human CD8 Clone RPA-T8 (555369), PerCP-Cy?5.5-conjugated mouse anti-human CD45RA Clone HI100 (563429), FITC-conjugated mouse anti-human CD62L Clone DREG-56 (555543), phycoerythrin (PE)-conjugated mouse anti-human CD64 Clone 10.1 (558592), FITC-conjugated mouse anti-human CD107A Clone H4A3 (555800), FITC-conjugated mouse anti-human IgG Clone G18-145 (555786), FITC-conjugated goat anti-mouse IgG (555988), FITC-conjugated anti-human CD279 (PD-1) clone MIH4 (557860), FITC-conjugated Annexin V (556420), propidium iodide (P1) staining solution (51-66211E), purified NA/LE mouse anti-human CD3 Clone UCHT1 (555329) and purified NA/LE mouse anti-Human CD28 Clone CD28.2 (555725) were purchased from BD Bioscience (San Jose, CA, USA). Anti-human B7-H3 (CD276) mAb 376.96 was developed and characterized as previously described. Anti-EGFR mAbs, cetuximab (Erbitux), and panitumumab (Vectibix) were obtained from Merck Serono (Darmstadt, Germany) and Amgen (Thousand Oaks, CA, USA), respectively. Human recombinant interleukin-7 (IL-7) (130-095-367) and interleukin-15 (IL-15) (130-095-760), human FcR blocking reagent (BR) (130-059-901), human CD14 microbeads (130-050-201), and human CD56 microbeads (130-050-401) were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Ficoll-Paque PLUS density gradient media was obtained from GE Healthcare Life science (Marlborough, MA, USA). (3-(4,5-Dimethylthiazol-2-Y1)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Sigma-Aldrich (Saint Louis, MO, USA). Retronectin (Recombinant Human Fibronectin) was purchased from Takara Bio (Saint-Germain-en-Laye, France).
Cell Lines
[0253] The 293T (RRID: CVCL_0063) packaging cell line was cultured in Iscove's Modified Dulbecco's Medium (IMDM). The KRAS-mutated HCT-116 (RRID: CVCL_0291) and firefly luciferase-expressing HCT-116 (HCT-116-Luc) cells were maintained in Roswell Park Memorial Institute (RPMI)-1640. The human CRC HT-29 (RRID: CVCL_0320) and Caco-2 (RRID: CVCL_0025) cell lines were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA). All mentioned media were supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 0.1 mg/ml streptomycin, and 100 U/ml penicillin to obtain cell culture complete media. BJ human skin fibroblasts (CVCL_3653) and human lung fibroblasts IMR-90 (CVCL_0347) were cultured in DMEM complete medium supplemented with 10% FBS (South American origin).). Human primary myoblasts were maintained in DMEM supplemented with primary skeletal muscle growth kit (ATCC? PCS-950-040TH). The 293T cells were provided by Dr. Gianpietro Dotti, University of North Carolina, Chapel Hill, NC, USA. The CRC HCT-116, HT-29, and Caco-2 cells were provided by Dr. Giulio Cesare Spagnoli, University of Basel, Switzerland. HCT-116-Luc cells were purchased from Caliper Life Sciences (Caliper Life Sciences, Inc., Hopkinton, MA, USA). The cell lines were passaged twice for a week and kept in culture for a maximum of 6-8 weeks.
CD64-CR and B7-H3 Constructs
[0254] Three CD64 chimeric receptors (CRs) were designed. The molecules were synthesized by GeneArt Gene Synthesis service (Thermo Fisher Scientific, Waltham, MA, USA). Table 1 describes the leader sequences and the domains chosen for assembling the CD64-CRs. The amino acid sequence of the molcules is shown in
TABLE-US-00013 TABLE 1 Leader Sequence Extracellular Transmembrane Intracellular Construct name (LS) Domain (Ec) Domain (Tm) domain CD64(Ec-Tm).28.? Human CD64 CD64 CD64 CD28 CD3? Hu-LS-CD64.28.? Human CD64 CD64 CD28 CD28 CD3? Ms-LS-CD64.28.? Murine CD64 CD64 CD28 CD28 CD3?
[0255] After synthesis, the gene cassettes of the chimer receptors were cloned into the NcoI and MluI sites of the SFG retroviral vector.
[0256] B7-H3.CAR, kindly provided by Gianpietro Dotti (UNC), was composed of scFv of the human B7-H3 mAb clone 376.96 fused to the transmembrane domain of CD8? and the intracellular motif of the CD28 and CD3? chain.
[0257] Amino acid sequences of selected CD64-CR constructs were synthesized for these studies as follows.
TABLE-US-00014 (MS-SP)-CD64.28.? Theaminoacidsequenceof(MS-SP)-CD64.28.?is: (SEQIDNO:1) MGILTSFGDDMWLLTTLLLWVPVGGQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQW FLNGTATQTSTPSYRITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWK DKLVYNVLYYRNGKAFKFFHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISVTVKELFPAPVLNASVTS PLLEGNLVTLSCETKLLLQRPGLQLYFSFYMGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLKR SPELELQVLGLQLPTPVWFHPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRR PGPTRKHYQPYAPPRDFAAYRSVDRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEM GGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD64.28.?, TheaminoacidsequenceofCD64.28.?is: (SEQIDNO:8) MGFLTTLLLWVPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTATQTS TPSYRITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWKDKLVYNVLYY RNGKAFKFFHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISVTVKELFPAPVLNASVTSPLLEGNLVTLS CETKLLLQRPGLQLYFSFYMGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLKRSPELELQVLGL QLPTPVWFHPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQP YAPPRDFAAYRSVDRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* CD64(EC-TM).28.? TheaminoacidsequenceofCD64(EC-TM).28.?is: (SEQIDNO:15) MGFLTTLLLWVPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTATQTS TPSYRITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWKDKLVYNVLYY RNGKAFKFFHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISVTVKELFPAPVLNASVTSPLLEGNLVTLS CETKLLLQRPGLQLYFSFYMGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLKRSPELELQVLGL QLPTPVWFHVLFYLAVGIMFLVNTVLWVTIVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYR SVDRVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*
[0258] In each of the above, the components are as follows:
TABLE-US-00015 (1)EXTRACELLULARREGIONOFTHEHUMANCD64 (SEQIDNO:3) (QVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTATQ TSTPSYRITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVF TEGEPLALRCHAWKDKLVYNVLYYRNGKAFKFFHWNSNLTILKTNISHNG TYHCSGMGKHRYTSAGISVTVKELFPAPVLNASVTSPLLEGNLVTLSCET KLLLQRPGLQLYFSFYMGSKTLRGRNTSSEYQILTARREDSGLYWCEAAT EDGNVLKRSPELELQVLGLQLPTPVWFH); (2)6aminoacidsfromCD28extracellularregion (SEQIDNO:4) (PGPSKP); (3)CD28TRANSMEMBRANE (SEQIDNO:5) (FWVLVVVGGVLACYSLLVTVAFIIFWV); (4)CD28CYTOPLASMICDOMAIN (SEQIDNO:6) (RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS); (5)VD:restrictionenzymessite; (6)CD35INTRACELLULARMOTIF (SEQIDNO:7) (RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKD TYDALHMQALPPR); (7)THESPDERIVEDFROMMOUSECD64(MS-SP) (SEQIDNO:2) (MGILTSFGDDMWLLTTLLLWVPVGG); (8)SPDERIVEDFROMHUMANCD64(HU-SP) (SEQIDNO:9) (MGFLTTLLLWVPVDG); and (9)CD64TRANSMEMBRANEREGION (SEQIDNO:18) (VLFYLAVGIMFLVNTVLWVTI)
[0259] The sequence identifiers for the foregoing sequences are shown in Table 2.
TABLE-US-00016 TABLE2 SequenceListing SEQID Construct/Component Sequence 1 (MS-SP)-CD64.28.? MGILTSFGDDMWLLTTLLLWVPVGGQVDTTKAVITLQPPWVSVFQE ETVTLHCEVLHLPGSSSTQWFLNGTATQTSTPSYRITSASVNDSGE YRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAW KDKLVYNVLYYRNGKAFKFFHWNSNLTILKTNISHNGTYHCSGMGK HRYTSAGISVTVKELFPAPVLNASVTSPLLEGNLVTLSCETKLLLQR PGLQLYFSFYMGSKTLRGRNTSSEYQILTARREDSGLYWCEAATE DGNVLKRSPELELQVLGLQLPTPVWFHPGPSKPFWVLVVVGGVLA CYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYA PPRDFAAYRSVDRVKFSRSADAPAYQQGQNQLYNELNLGRREEY DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 2 SPDERIVEDFROM MGILTSFGDDMWLLTTLLLWVPVGG MOUSECD64(MS-SP) 3 EXTRACELLULAR QVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGT REGIONOFTHE ATQTSTPSYRITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLL HUMANCD64 QVSSRVFTEGEPLALRCHAWKDKLVYNVLYYRNGKAFKFFHWNSN LTILKTNISHNGTYHCSGMGKHRYTSAGISVTVKELFPAPVLNASVT SPLLEGNLVTLSCETKLLLQRPGLQLYFSFYMGSKTLRGRNTSSEY QILTARREDSGLYWCEAATEDGNVLKRSPELELQVLGLQLPTPVWFH 4 6AMINOACIDS PGPSKP FROMCD28 EXTRACELLULAR REGION 5 CD28 FWVLVVVGGVLACYSLLVTVAFIIFWV TRANSMEMBRANE 6 CD28CYTOPLASMIC RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS DOMAIN 7 CD35 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPE INTRACELLULAR MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD MOTIF GLYQGLSTATKDTYDALHMQALPPR 8 CD64.28.?, MGFLTTLLLWVPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEVL HLPGSSSTQWFLNGTATQTSTPSYRITSASVNDSGEYRCQRGLSG RSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWKDKLVYNVLY YRNGKAFKFFHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISV TVKELFPAPVLNASVTSPLLEGNLVTLSCETKLLLQRPGLQLYFSFY MGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLKRSPE LELQVLGLQLPTPVWFHPGPSKPFWVLVVVGGVLACYSLLVTVAFII FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS VDRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRD PEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR* 9 SPDERIVEDFROM MGFLTTLLLWVPVDG HUMANCD64(HU-SP) 10 EXTRACELLULAR QVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGT REGIONOFTHE ATQTSTPSYRITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLL HUMANCD64 QVSSRVFTEGEPLALRCHAWKDKLVYNVLYYRNGKAFKFFHWNSN LTILKTNISHNGTYHCSGMGKHRYTSAGISVTVKELFPAPVLNASVT SPLLEGNLVTLSCETKLLLQRPGLQLYFSFYMGSKTLRGRNTSSEY QILTARREDSGLYWCEAATEDGNVLKRSPELELQVLGLQLPTPVWF H 11 6AMINOACIDS PGPSKP FROMCD28 EXTRACELLULAR REGION 12 CD28 FWVLVVVGGVLACYSLLVTVAFIIFWV TRANSMEMBRANE 13 CD28CYTOPLASMIC RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS DOMAIN 14 CD3? RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPE INTRACELLULAR MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD MOTIF GLYQGLSTATKDTYDALHMQALPPR 15 CD64(EC-TM).28.? MGFLTTLLLWVPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEVL HLPGSSSTQWFLNGTATQTSTPSYRITSASVNDSGEYRCQRGLSG RSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWKDKLVYNVLY YRNGKAFKFFHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISV TVKELFPAPVLNASVTSPLLEGNLVTLSCETKLLLQRPGLQLYFSFY MGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLKRSPE LELQVLGLQLPTPVWFHVLFYLAVGIMFLVNTVLWVTIVRSKRSRLL HSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSVDRVKFSRSAE PPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKN PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT KDTYDALHMQALPPR* 16 SPDERIVEDFROM MGFLTTLLLWVPVDG HUMANCD64(HU-SP) 17 EXTRACELLULAR QVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGT REGIONOFTHE ATQTSTPSYRITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLL HUMANCD64 QVSSRVFTEGEPLALRCHAWKDKLVYNVLYYRNGKAFKFFHWNSN LTILKTNISHNGTYHCSGMGKHRYTSAGISVTVKELFPAPVLNASVT SPLLEGNLVTLSCETKLLLQRPGLQLYFSFYMGSKTLRGRNTSSEY QILTARREDSGLYWCEAATEDGNVLKRSPELELQVLGLQLPTPVWF H 18 CD64 VLFYLAVGIMFLVNTVLWVTI TRANSMEMBRANE REGION 19 CD28CYTOPLASMIC RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS DOMAIN 20 CD3? RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPE INTRACELLULAR MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD MOTIF GLYQGLSTATKDTYDALHMQALPPR 21 FULL-LENGTH MWFLTTLLLWVPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEVL HUMANCD64 HLPGSSSTQWFLNGTATQTSTPSYRITSASVNDSGEYRCQRGLSG (UNIPROT RSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWKDKLVYNVLY ACCESSIONNO. YRNGKAFKFFHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISV P12314-1) TVKELFPAPVLNASVTSPLLEGNLVTLSCETKLLLQRPGLQLYFSFY MGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLKRSPE LELQVLGLQLPTPVWFHVLFYLAVGIMFLVNTVLWVTIRKELKRKKK WDLEISLDSGHEKKVISSLQEDRHLEEELKCQEQKEEQLQEGVHRK EPQGAT 22 CD64 VLFYLAVGIMFLVNTVLWVTIV TRANSMEMBRANE DOMAINWITHA VALINE 23 CD28ANDCD3-ZETA RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSVDR SIGNALINGDOMAIN VKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEM GGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL YQGLSTATKDTYDALHMQALPPR
Retrovirus Production
[0260] Retroviral supernatant production was performed as previously described.sup.28,29.
[0261] Briefly, the 293T packaging cells were cotransfected with CD64-CR construct plus the Peg-Pam and the RDF vectors encoding MoMLV gag-pol proteins and RD114 envelope, respectively. Cell supernatant containing retroviral particles were harvested 48 h and 72 h after transfection. RetroNectin-coated non-tissue culture treated 24-well plates were preloaded with 1 ml/well of viral supernatant and used for T cell transduction.
PBMCs Isolation and Human T Cell Transduction
[0262] Blood samples were obtained from the Department of Transfusion Medicine of the Tor Vergata Hospital, Rome. Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats with Ficoll-paque Plus reagent according to the manufacturer's instruction and CD56.sup.+ NK cells were removed utilizing the human CD56 microbeads. For activation, T cells were cultured for 72 hours in a non-tissue culture treated 24-well plate precoated with 1 ?g/ml of anti-human CD3 and 1 ?g/ml of anti-human CD28 mAb mix. Then, proliferating T cells (0.5?10.sup.6/well) were seeded into a retroviral-preloaded 24-well plate for 72 h at 37? C. in 5% CO.sub.2. After transduction, the T cells were expanded in RPMI-1640 complete medium supplemented with 10 ng/ml of IL-7 and 5 ng/ml of IL-15 for further analysis. All the experiments were conducted without the addition of the cytokine to the medium.
Magnetic Sorting of CD14+ Cells
[0263] CD14+ monocytes were selected from PBMCs using human CD14 microbeads (Miltenyi) according to the manufacturers instruction. Briefly, a pellet of 30?10.sup.6 PBMCs was incubated with 60 ?l of anti-human CD14 microbeads for 15 min at 4? C. Then, the stained cells were flowed through a BS column (Miltenyi) for a positive selection. Then CD14+ adherent cells were eluted by mechanical pressure. The average purity of the recovered CD14+ cells was higher than 95%.
Binding Assay of the Monoclonal Antibody Fc on the CD64-CR
[0264] CD64-CR transduced T cells were pre-incubated for 15 min at 4? C. with or without 54 of FcR blocking reagent (FcR BR) and then incubated with 10 ?g/ml of the anti-human EGFR cetuximab (IgG1), panitumumab (IgG2) or mouse anti-human B7-H3 mAb (376.96) (IgG2A) for 30 min at 4? C. Then, cells were washed and stained with FITC-conjugated mouse anti-human IgG or FITC-conjugated goat anti-mouse IgG for 30 min at 4? C. In dose-response experiments, the cells were incubated for 30 min at 4? C. with increasing amounts of cetuximab, panitumumab, and 376.96 mAb and then stained with the above-mentioned secondary antibodies. In the competition test, the CD64-CR T cells were incubated with different doses of heat-inactivated human plasma for 30 min at 4? C. or 37? C. and then 10 ?g/ml of 376.96 mAb was added for 30 min at 4? C. or 37? C. The analysis of the mAb binding on the CD64-CR T cell surface was performed by flow cytometry.
Flow Cytometry
[0265] The assessment of CD64-CR expression on T cells and phenotypic analysis of transduced T cell subsets was performed by flow cytometry. After transduction, T cells were incubated for 30 min at 4? C. with mAbs specific for CD3, CD64, CD45RA, CD62L, CCR7 conjugated with FITC, PE, PerCP-Cy5.5, FITC, and APC fluorochromes, respectively. After staining, cells were washed and fixed with 1% of the formaldehyde-PBS solution. Samples were acquired with a 2-laser BD FACSCalibur? flow cytometer using BD CellQuest software (Becton Dickinson, Franklin Lakes, NJ, USA). All data were analyzed using Tree Star, Inc. FlowJo software.
In Vitro Coculture Experiments
[0266] MTT assay. CRC or non-tumor target cells (7?10.sup.3) were cocultured into flat-bottom 96-well plates with CD64-CR T cells at different effector: target (E:T) ratios in the presence or absence of 1 ?g/ml of cetuximab or anti-human B7-H3 (376.96) mAb, for 72 hours at 37? C. An experimental triplicate was performed for each condition. After incubation, target cell viability was determined by MTT assay as previously detailed (Arriga et al., Int J Cancer 2020; 146:2531-8; Caratelli et al., Int J Cancer 2020; 146:236-47).
[0267] Bioluminescent Imaging (BLI). HCT-116-luc cells were seeded in 96-well microplates in triplicate (n=3) and incubated with transduced or non-transduced T cells at various E:T ratios. After 3-day coculture, the cell culture medium was supplemented with D-luciferin (PerkinElmer) dissolved in PBS (150 ?g/mL) for 10 min and analysis was performed using the IVIS? Lumina II platform (PerkinElmer, Waltham, MA, USA). Photons emitted from luciferase-expressing cells in selected regions of interest (ROI) were quantified using the Living Image? software.
[0268] Target Cell depletion. HCT-116, HT-29, and CaCo-2 CRC cell lines (0.02?10.sup.6/well) were cocultured in triplicate in 96-well plate with CD64-CR transduced or non-transduced control T cells at various E:T ratio for approximately 18 hours. Then, non-adherent effector T cells were removed and the remaining adherent CRC target cells were enumerated by trypan blue-exclusion cell count.
[0269] Flow-cytometry cytotoxicity assay. HCT-116 target cells (0.2?10.sup.6) were seeded in a 24-well plate. After 6-hour incubation, 0.4?10.sup.6 CD64-CR transduced or non-transduced T cells (E:T=2:1) were added to the wells with or without 1 ?g/ml of cetuximab or 376.96 mAb for approximately 18 hours at 37? C. To evaluate the target cell death levels, the cell coculture were collected and stained with APC-conjugated mouse anti-human CD3, FITC-conjugated annexin V and PI solution for 15 min at room temperature, in the dark. The percentage of target cell apoptosis/necrosis was determined by flow cytometry.
CD107A Release Test
[0270] CD64-CR T cells (0.4?10.sup.6) were incubated with HCT116 (0.2?10.sup.6) cells in 96-well microplates in the presence or absence of 1 ?g/ml of cetuximab, panitumumab, 376.96. To maximize CD107A detection, FITC-conjugated mouse anti-human CD107A mAb was included during coculture with the addition of 2 ?M of the secretion inhibitor monensin used for blocking CD107A antibody acidification and degradation. After a 4-hour-incubation at 37? C., cells were stained with PE-conjugated mouse anti-human CD64 and analyzed by flow cytometry.
Immunofluorescence and Confocal Microscopy
[0271] HCT116 colon cancer cells were cocultured with CD64-CR expressing T cells for 30 min, fixed for 10 minutes with 3% (w/v) formaldehyde in PBS, permeabilized 10 minutes with 0.15% Triton, blocked for 1 hr 5% BSA, and immunostained with anti-EGFR (1:20, cell signaling 08/2019 D3861), anti-CD64 (1:40, BD 555525), overnight at 4? C. in 1% BSA. Cells were incubated with the following secondary antibodies (diluted in 1% BSA) at room temperature for 1 h: Cy3-conjugated donkey anti-mouse and Cy5-conjugated donkey anti-rabbit antibodies (Jackson ImmunoResearch Laboratories). Then, cells were immunostained 2 h at 37? C. with FITC conjugated anti-human perforin antibody (1:10). The DNA was counterstained with 0.4 mg/ml DAPI (33258; Sigma). Slides were mounted in 50% glycerol and analyzed within 24 h. Images were recorded by using a Zeiss LSM 880 confocal laser scanning microscope equipped with a 60X/1.23 NA oil immersion objective, and laser 405, 488, 543, 633 nm were used to excite the fluorophores.
Statistical Analysis
[0272] Data sets were analyzed using Graphpad Prism Software. Unpaired two-tailed T-test and two-way ANOVA tests were used. Differences with a p-value <0.05 were considered to be statistically significant.
Uses
[0273] The human Fc receptor CD64, as described herein, has been linked to the stimulatory molecules CD28 and CD3. This construct is transduced to human T cells. The CD64 T cells in combination with the B7-H3-specific mAb 376.96 acquire the ability to specifically eliminate human tumor cells expressing B7-H3. Therefore CD64 T cells in combination with mAb 376.96 are useful for immunotherapy of tumors which express B7-H3. This combination can be extended to other tumor antigen-specific mAbs. Table 3 shows an exemplary list cancer cell lines tested positive for B7-H3 antigen.
TABLE-US-00017 TABLE 3 EXEMPLARY LIST OF B7-H3 ANTIGEN EXPRESSING CELLS U937: AML ML-2: AML KMN-1: MULTIPLE MYELOMA HCT-116: COLORECTAL CARCINOMA HT-29: COLORECTAL CARCINOMA MDA-MB-231: BREAST CARCINOMA MDA-MB-468: BREAST CARCINOMA A549: NON SMALL CELL LUNG CARCINOMA U87: GLIOBLASTOMA MULTIFORME (GB) U373: GB CSC2: GB (CANCER STEM CELLS) CSC3: GB (CANCER STEM CELLS) FADU: HEAD AND NECK CANCER A-253: HEAD AND NECK CANCER
[0274] Nucleic acids encoding any of the CRs described herein are useful in the invention, including the FcgRI (CD64) chimeric receptor or a binding domain thereof (such as the Fc?RI D2 domain C-strand, C-strand, and/or the CE loop) resulting from the fusion of the extracellular or extracellular plus transmembrane domain of CD64 with a T cell costimulatory molecule joint to the T cell receptor chain. This molecule is cloned into an expression vector that is transduced or transfected in a host cell in combination with the 396.96 monoclonal antibody (mAb) with specificity for the B7-H3 antigen for immunotherapeutic applications including cancer and other diseases.
[0275] The invention has many applications in the field of cancer therapy. Products described herein are useful for the implementation of adoptive T cell transfer-based immunotherapies for the treatment of a wide range of hematologic and solid B7-H3 positive tumors (such as colorectal cancer, breast cancer, non-small cell lung cancer, head and neck cancer, glioblastoma, leukemia, myeloma).
OTHER EMBODIMENTS
[0276] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.
[0277] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations described herein following, in general, the principles described herein and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.
[0278] Other embodiments are within the claims.