Compositions and methods for treating egfr positive cancers

Abstract

The disclosure provides immune cells comprising a first activator receptor and a second inhibitory receptor, and methods of making and using same for the treatment of cancer.

What is claimed is: 
     
         1 . An immune cell, comprising:
 a. a first receptor, comprising an extracellular ligand binding domain specific to Epidermal Growth Factor Receptor (EGFR); and   b. a second receptor, comprising an extracellular ligand binding domain specific to a non-target antigen lost in EGFR+ cancer due to loss of heterozygosity,   wherein the first receptor is an activator receptor responsive to EGFR; and wherein the second receptor is an inhibitory receptor responsive to the non-target antigen.   
     
     
         2 . The immune cell of  claim 1 , wherein the extracellular ligand binding domain of the second receptor specifically binds an allelic variant of an MHC protein. 
     
     
         3 . The immune cell of  claim 1 , wherein the extracellular ligand binding domain of the second receptor specifically binds an allelic variant of an HLA-A, HLA-B, or HLA-C protein. 
     
     
         4 . The immune cell of  claim 1 , wherein the extracellular ligand binding domain of the second receptor specifically binds HLA-A*02. 
     
     
         5 . The immune cell of  claim 1 , wherein the extracellular ligand binding domain of the second receptor specifically binds HLA-A*01, HLA-A*03, HLA-A*11, HLA-C*07, or HLA-B*07. 
     
     
         6 . The immune cell of any one of  claims 3 - 5 , wherein the extracellular ligand binding domain of the second receptor comprises complementarity determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 as disclosed Table 5; or CDR sequences having at most 1, 2, or 3 substitutions, deletions, or insertion relative to the CDRs of Table 5. 
     
     
         7 . The immune cell of any one of  claims 3 - 5 , wherein the extracellular ligand binding domain of the second receptor comprises complementarity determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 of SEQ ID NOs: 101-106 or of SEQ ID NOs: 106-112; or CDR sequences having at most 1, 2, or 3 substitutions, deletions, or insertion relative to the CDRs of Table 5. 
     
     
         8 . The immune cell of any one of  claims 3 - 5 , wherein the extracellular ligand binding domain of the second receptor comprises a polypeptide sequence selected from the polypeptide sequence disclosed in Table 4; or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto. 
     
     
         9 . The immune cell of  claim 4 , wherein the extracellular ligand binding domain of the second receptor comprises any one of SEQ ID NOs: 89-100; or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto. 
     
     
         10 . The immune cell of any one of  claims 1 - 9 , wherein the first receptor is a chimeric antigen receptor (CAR). 
     
     
         11 . The immune cell of any one of  claims 1 - 10 , wherein the extracellular ligand binding domain of the first receptor comprises a variable heavy (VH) portion comprising a set of heavy chain complementarity determining regions (HC-CDRs) selected from the group of sequences set forth Table 3; and/or a variable light (VL) portion comprising a set of light chain complementarity determining regions (LC-CDRs) from the group of sequences set forth in Table 3; or CDR sequences having at most 1, 2, 3, 4 substitutions, insertions, or deletions in each CDR. 
     
     
         12 . The immune cell of any one of  claims 1 - 11 , wherein the extracellular ligand binding domain of the first receptor comprises a variable heavy (VH) portion having a sequence selected from the VH sequence set forth in Table 2; and/or a variable light (VL) portion comprising a sequence set forth in Table 2; or sequences having at least 70%, at least 85%, at least 90%, or at least 95% identity thereto. 
     
     
         13 . The immune cell of any one of  claims 1 - 12 , wherein the extracellular ligand binding domain of the first receptor comprising a sequence selected from the group of sequences set forth in Table 1; or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. 
     
     
         14 . The immune cell of any one of  claims 1 - 13 , wherein the extracellular ligand binding domain of the first receptor comprises an scFv sequence selected from the group consisting of SEQ ID NO: 9-18; or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto. 
     
     
         15 . The immune cell of any one of  claims 1 - 14 , wherein the second receptor comprises a LILRB1 intracellular domain or a functional variant thereof. 
     
     
         16 . The immune cell of  claim 15 , wherein the LILRB1 intracellular domain comprises a sequence at least 90%, at least 95%, at least 97%, at least 99%, or is identical to SEQ ID NO: 129. 
     
     
         17 . The immune cell of any one of  claims 1 - 16 , wherein the second receptor comprises a LILRB1 transmembrane domain or a functional variant thereof. 
     
     
         18 . The immune cell of  claim 17 , wherein the LILRB1 transmembrane domain or a functional variant thereof comprises a sequence at least 90%, at least 95%, at least 97%, at least 99% or is identical to SEQ ID NO: 133. 
     
     
         19 . The immune cell of any one of  claims 1 - 18 , wherein the second receptor comprises a LILRB1 hinge domain or functional variant thereof. 
     
     
         20 . The immune cell of  claim 19 , wherein the LILRB1 hinge domain comprises a sequence at least 90%, at least 95%, at least 97%, at least 99% or is identical to SEQ ID NO: 132, SEQ ID NO: 125, SEQ ID NO: 126. 
     
     
         21 . The immune cell of any one of  claims 1 - 20 , wherein the second receptor comprises a LILRB1 intracellular domain, a LILRB1 transmembrane domain, a LILRB1 hinge domain, a functional variant of any of these, or combinations thereof. 
     
     
         22 . The immune cell of  claim 21 , wherein the LILRB1 intracellular domain and LILRB1 transmembrane domain comprises SEQ ID NO: 128 or a sequence at least 95% identical to SEQ ID NO: 128. 
     
     
         23 . The immune cell of any one of  claims 1 - 22 , wherein the EGFR+ cancer cell is a lung cancer cell, a small cell lung cancer cell, a non-small cell lung cancer cell, a pancreatic ductal carcinoma cell, a colorectal cancer cell, a head and neck cancer cell, a esophagus and gastric adenocarcinoma cell, an ovarian cancer cell, a glioblastoma multiforme cell, a cervical squamous cell carcinoma cell, a kidney cancer cell, a papillary kidney cancer cell, a kidney renal clear cell carcinoma cell, a bladder cancer cell, a breast cancer cell, a bile duct cancer cell, a liver cancer cell, a prostate cancer cell, a sarcoma cell, a thyroid cancer cell, a thymus cancer cell, a stomach cancer cell, or a uterine cancer cell. 
     
     
         24 . The immune cell of  claim 23 , wherein the EGFR+ cancer cell is a lung cancer cell. 
     
     
         25 . The immune cell of any one of  claims 1 - 24 , wherein the EGFR+ cancer cell is an EGFR+/HLA-A*02− cancer cell that does not express HLA-A*02; or a cancer cell derived from an HLA-A*02+ individual which does not express HLA-A*02. 
     
     
         26 . The immune cell of  claim 25 , wherein the EGFR+/HLA-A*02− cancer cell is derived from an EGFR+/HLA-A*02+ cell by loss of heterozygosity at HLA-A leading to loss of HLA-A*02. 
     
     
         27 . The immune cell of any one of  claims 1 - 26 , wherein the first receptor and the second receptor together specifically activate the immune cell in the presence of the EGFR/HLA-A*02− cancer cell having loss of heterozygosity. 
     
     
         28 . The immune cell of any one of  claims 1 - 27 , wherein the first receptor and the second receptor together do not specifically activate the immune cell in the presence of an EGFR+ cell that has not lost HLA-A*02 by loss of heterozygosity. 
     
     
         29 . The immune cell of any one of  claims 1 - 28 , wherein the immune cell is a T cell, macrophage, NK cell, iNKT cell, or a gamma delta T cell. 
     
     
         30 . The immune cell of  claim 29 , wherein the T cell is a CD8+CD4− T cell. 
     
     
         31 . The immune cell of any one of  claims 1 - 30 , wherein expression and/or function of an MHC Class I gene has been reduced or eliminated. 
     
     
         32 . The immune cell of  claim 30 , wherein the MHC Class I gene is beta-2-microglobulin (B2M). 
     
     
         33 . The immune cell of  claim 32 , further comprising a polynucleotide comprising an interfering RNA, the interfering RNA comprising a sequence complementary to a sequence of a B2M mRNA (SEQ ID NO: 172). 
     
     
         34 . The immune cell of  claim 33 , wherein the interfering RNA is capable of inducing RNAi-mediated degradation of the B2M mRNA. 
     
     
         35 . The immune cell of  claim 33 , wherein the interfering RNA is a short hairpin RNA (shRNA). 
     
     
         36 . The immune cell of  claim 35 , wherein the shRNA comprises:
 a. A first sequence, having from 5′ end to 3′ end a sequence complementary to a sequence of the B2M mRNA; and   b. A second sequence, having from 5′ end to 3′ end a sequence complementary to the first sequence,   wherein the first sequence and the second sequence form the shRNA.   
     
     
         37 . The immune cell of  claim 32 , comprising one or more modifications to a sequence encoding B2M (SEQ ID NO: 170), wherein the one or more modifications reduce the expression and/or eliminate the function of B2M. 
     
     
         38 . The immune cell of  claim 37 , wherein the one or more modifications comprise one or more inactivating mutations of the endogenous gene encoding B2M. 
     
     
         39 . The immune cell of  claim 38 , wherein the one or more inactivating mutations comprise a deletion, an insertion, a substitution, or a frameshift mutation. 
     
     
         40 . The immune cell of any one of  claims 38 - 39 , wherein the one or more inactivating mutations are introduced with a nucleic acid guided endonuclease in a complex with at least one guide nucleic acid (gNA) that specifically targets a sequence of the endogenous gene encoding B2M (SEQ ID NO: 170). 
     
     
         41 . The immune cell of  claim 31 , wherein the MHC Class I gene is HLA-A*02. 
     
     
         42 . The immune cell of  claim 41 , further comprising a polynucleotide comprising an interfering RNA, comprising a sequence complementary to a sequence of an HLA-A*02 mRNA (SEQ ID NO: 171). 
     
     
         43 . The immune cell of  claim 42 , wherein the interfering RNA is capable of inducing RNA interference (RNAi)-mediated degradation of the HLA-A*02 mRNA. 
     
     
         44 . The immune cell of  claim 43 , wherein the interfering RNA is a short hairpin RNA (shRNA) comprising:
 a. a first sequence, having from 5′ end to 3′ end a sequence complementary to a sequence of the HLA-A*02 mRNA; and   b. a second sequence, having from 5′ end to 3′ end a sequence complementary to the first sequence,   wherein the first sequence and the second sequence form the shRNA.   
     
     
         45 . The immune cell of  claim 41 , comprising one or more modifications to a sequence of an endogenous gene encoding HLA-A*02 (SEQ ID NO: 169), wherein the one or modifications reduce the expression and/or eliminate the function of HLA-A*02. 
     
     
         46 . The immune cell of  claim 45 , wherein the one or more modifications comprise one or more inactivating mutations of the endogenous gene encoding HLA-A*02. 
     
     
         47 . The immune cell of  claim 45  or  claim 46 , wherein the one or more inactivating mutations are introduced with a nucleic acid guided endonuclease in a complex with at least one guide nucleic acid (gNA) that specifically targets a sequence of the endogenous gene encoding HLA-A*02. 
     
     
         48 . The immune cell of any one of  claims 1 - 47 , wherein:
 a. the first receptor comprises SEQ ID NO: 177, or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and   b. the second receptor comprises SEQ ID NO: 174 or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.   
     
     
         49 . The immune cell of any one of  claims 1 - 47 , wherein:
 a. the first receptor comprises SEQ ID NO: 177, or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and   b. the second receptor comprises SEQ ID NO: 173, or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.   
     
     
         50 . The immune cell of any one of  claims 1 - 47 , wherein:
 a. the first receptor comprises SEQ ID NO: 175, or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and   b. the second receptor comprises SEQ ID NO: 174, or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.   
     
     
         51 . The immune cell of any one of  claims 1 - 47 , wherein:
 a. the first receptor comprises SEQ ID NO: 175, or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and   b. the second receptor comprises SEQ ID NO: 173, or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.   
     
     
         52 . The immune cell of any one of  claims 1 - 47 , wherein:
 a. the first receptor comprises SEQ ID NO: 176, or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and   b. the second receptor comprises SEQ ID NO: 173, or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.   
     
     
         53 . The immune cell of any one of  claims 1 - 47 , wherein:
 a. the first receptor comprises SEQ ID NO: 176, or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and   b. the second receptor comprises SEQ ID NO: 173, or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.   
     
     
         54 . The immune cell of any one of  claims 1 - 53 , further comprising a T2A self-cleaving peptide, wherein the T2A self-cleaving peptide comprises SEQ ID NO: 178, or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto. 
     
     
         55 . The immune cell of any one of  claims 1 - 54 , further comprising an interfering RNA, wherein the interfering RNA comprises SEQ ID NO: 179, or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto. 
     
     
         56 . The immune cell of any one of  claims 1 - 55 , wherein the immune cell is autologous. 
     
     
         57 . The immune cell of any one of  claims 1 - 55 , wherein the immune cell is allogeneic. 
     
     
         58 . A pharmaceutical composition, comprising a therapeutically effective amount of the immune cells of any one of  claims 1 - 57 . 
     
     
         59 . The pharmaceutical composition of  claim 58 , wherein the immune cell expresses both the first receptor and the second receptor. 
     
     
         60 . The pharmaceutical composition of  claim 59 , wherein at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the immune cells express both the first receptor and the second receptor. 
     
     
         61 . The pharmaceutical composition of  claim 59 , wherein at least 90% of the immune cells express both the first receptor and the second receptor. 
     
     
         62 . The pharmaceutical composition of any one of  claims 58 - 61 , further comprising a pharmaceutically acceptable carrier, diluent or excipient. 
     
     
         63 . The pharmaceutical composition of any one of  claims 58 - 62 , for use as a medicament in the treatment of EGFR+ cancer. 
     
     
         64 . A polynucleotide or polynucleotide system, comprising one or more polynucleotides comprising polynucleotide sequences encoding:
 a. a first receptor, comprising an extracellular ligand binding domain specific to Endothelial Growth Factor Receptor (EGFR); and   b. a second receptor, comprising an extracellular ligand binding domain specific to a non-target antigen that has been lost in the EGFR+ cancer cell due to loss of heterozygosity,   wherein the first receptor is an activator receptor responsive to EGFR on the EGFR+ cancer cell; and wherein the second receptor is an inhibitory receptor responsive to the non-target antigen.   
     
     
         65 . A polynucleotide or polynucleotide system, comprising one or more polynucleotides comprising polynucleotide sequences encoding the first receptor and the second receptor for use in generating the immune cells of any one of  claims 1 - 57 . 
     
     
         66 . A vector, comprising the one or more polynucleotides of  claim 64  or  65 . 
     
     
         67 . A method of killing EGFR+ cancer cell having loss of heterozygosity at an MHC class I locus, comprising administering to a subject an effective amount of the immune cell of any one of  claims 1 - 57  or the pharmaceutical composition of any one of  claims 58 - 62 . 
     
     
         68 . The method of  claim 67 , wherein the EGFR+ cancer cell is a lung cancer cell, a small cell lung cancer cell, a non-small cell lung cancer cell, a pancreatic ductal carcinoma cell, a colorectal cancer cell, a head and neck cancer cell, a esophagus and gastric adenocarcinoma cell, an ovarian cancer cell, a glioblastoma multiforme cell, a cervical squamous cell carcinoma cell, a kidney cancer cell, a papillary kidney cancer cell, a kidney renal clear cell carcinoma cell, a bladder cancer cell, a breast cancer cell, a bile duct cancer cell, a liver cancer cell, a prostate cancer cell, a sarcoma cell, a thyroid cancer cell, a thymus cancer cell, a stomach cancer cell, or a uterine cancer cell. 
     
     
         69 . The method of  claim 67 , wherein the EGFR+ cancer cell is a lung cancer cell. 
     
     
         70 . The method of  claim 67 , wherein the EGFR+ cancer cell is an EGFR+/HLA-A*02− cancer cell that does not express HLA-A*02; or a cancer cell derived from an HLA-A*02+ individual which does not express HLA-A*02. 
     
     
         71 . The method of  claim 70 , wherein the EGFR+/HLA-A*02− cancer cell is derived from an EGFR+/HLA-A*02+ cell by loss of heterozygosity at HLA-A leading to loss of HLA-A*02. 
     
     
         72 . A method of treating EGFR+ cancer in a subject having an EGFR+ tumor having loss of heterozygosity at a locus encoding a non-target antigen, comprising administering to the subject an effective amount of the immune cell of any one of  claims 1 - 57  or the pharmaceutical composition of any one of  claims 58 - 62 . 
     
     
         73 . The method of  claim 72 , wherein the subject is a heterozygous HLA-A*02 patient with a malignancy that expresses EGFR and has lost HLA-A*02 expression. 
     
     
         74 . The method of  claim 72 , wherein the subject is a heterozygous HLA-A*02 patient with recurrent unresectable or metastatic solid tumors that express EGFR and have lost HLA-A*02 expression. 
     
     
         75 . A method of treating a cancer in a subject comprising:
 a. determining the genotype or expression level of a non-target antigen in non-malignant cells and cancer cells of the subject;   b. determining the expression level of EGFR in cancer cells of the subject; and   c. administering to the subject an effective amount of the immune cell of any one of  claims 1 - 57  or the pharmaceutical composition of any one of  claims 58 - 62  if the non-malignant cells express the non-target antigen and the cancer cells do not express the non-target antigen, and the cancer cells are EGFR positive.   
     
     
         76 . The method of any one of  claims 67 - 75 , wherein administration of the immune cell of any one of  claims 1 - 57  or the pharmaceutical composition of any one of  claims 58 - 62  reduces the size of a tumor in the subject. 
     
     
         77 . The method of  claim 76 , wherein the tumor is reduced by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%. 
     
     
         78 . The method of  claim 76 , wherein the tumor is eliminated. 
     
     
         79 . The method of any one of  claims 67 - 75 , wherein administration of the immune cell or the pharmaceutical composition arrests the growth of a tumor in the subject. 
     
     
         80 . The method of any one of  claims 67 - 75 , wherein administration of the immune cell of any one of  claims 1 - 57  or the pharmaceutical composition of any one of  claims 58 - 62  reduces the number of tumors in the subject. 
     
     
         81 . The method of any one of  claims 67 - 80 , wherein administration of the immune cell or the pharmaceutical composition results in selective killing of a cancer cell but not a normal cell in the subject. 
     
     
         82 . The method of  claim 81 , wherein at least about 60% of the cells killed are cancer cells, at least about 65% of the cells killed are cancer cells, at least about 70% of the cells killed are cancer cells, at least about 75% of the cells killed are cancer cells, at least about 80% of the cells killed are cancer cells, at least about 85% of the cells killed are cancer cells, at least about 90% of the cells killed are cancer cells, at least about 95% of the cells killed are cancer cells, or about 100% of the cells killed are cancer cells. 
     
     
         83 . The method of  claim 81 , wherein administration of the immune cell or pharmaceutical composition results in the killing of about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or all of the cancer cells of the subject. 
     
     
         84 . The method of any one of  claims 67 - 83 , wherein administration of the immune cell or the pharmaceutical composition results in fewer side effects for the subject than administration of an otherwise equivalent immune cell comprising the first activator receptor but no second inhibitory receptor. 
     
     
         85 . A method of making a plurality of immune cells, comprising:
 a. providing a plurality of immune cells, and   b. transforming the plurality of immune cells with the polynucleotide system of  claim 64  or  claim 65 , or the vector of  claim 66 .   
     
     
         86 . A kit comprising the immune cell of any one of  claims 1 - 57  or the pharmaceutical composition of any one of  claims 58 - 62 . 
     
     
         87 . The kit of  claim 86 , further comprising instructions for use.