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  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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  • C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
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  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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  • C07K14/7051—T-cell receptor (TcR)-CD3 complex
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  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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  • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
• • C—CHEMISTRY; METALLURGY
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  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/622—Single chain antibody (scFv)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
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• • C—CHEMISTRY; METALLURGY
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  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/033—Fusion polypeptide containing a localisation/targetting motif containing a motif for targeting to the internal surface of the plasma membrane, e.g. containing a myristoylation motif
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  • C12N2510/00—Genetically modified cells
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  • C12N2740/16011—Human Immunodeficiency Virus, HIV
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  • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

• This invention is directed to chimeric antigen receptors and cells comprising the same, wherein the cells further secrete monoclonal antibodies locally at a tumor site.
• Clear cell renal cell carcinoma is the major type of RCC which is among the 10 most common cancers in both men and women.
• Other types of renal carcinoma include papillary renal cell carcinoma, chromophobe renal cell carcinoma, and other or unclassified types of renal cell carcinoma. See, for example, Lancet 373 (2009) 1119-32.
• the chimeric antigen receptor comprises an extracellular ligand binding domain that is specific for a first antigen and a second antigen on the surface of a cancer cell, wherein the first antigen comprises CAIX and the second antigen comprises CD70.
• the CAR further comprises a transmembrane polypeptide, an intracellular signaling domain, and/or a co-stimulatory domain.
• the extracellular ligand binding domain comprises an antibody or fragment thereof.
• the antibody comprises a VH and/or VL according to Table 2, or any combination thereof.
• the antibody comprises a VH and/or VL according to Table 4, or any combination thereof.
• the extracellular binding domain comprises a a VH and/or a VL of Table 2 and Table 4, or any combination thereof.
• the antibody comprises a CDR1, CDR2, and/or CDR3 of Table 1, or any combination thereof.
• the antibody comprises a CDR1, CDR2, and/or CDR3 of Table 3, or any combination thereof.
• the extracellular binding domain comprises a CDR1, CDR2, and/or CDR3 of Table 1 and Table 3, or any combination thereof.
• the engineered cell expresses and secretes a recombinant polypeptide.
• the recombinant polypeptide comprises an antibody or fragment thereof, or a cytokine.
• the recombinant polypeptide comprises an antibody or fragment thereof specific for TIGIT, GITR, PD-L1, PD-L2, PD-1, CTLA-4, VISTA, CD70, TIM-3, LAG-3, CD40L, or CCR4.
• the recombinant polypeptide comprises a cytokine comprising IL-12, IL-15, or IL-18.
• the recombinant polypeptide modulates the immune system of a subject.
• the recombinant polypeptide is an immune checkpoint blockade antibody.
• the recombinant polypeptide modulates tumor vasculogenesis.
• the recombinant polypeptide can be specific for VEGF, VEGFR1, VEGFR2, PDGF, Ang-1, or ATI.
• the engineered cell is a T cell, an NK cell, or an NKT cell.
• the T cell is CD4+, CD8+, CD3+ panT cells, or any combination thereof.
• the T cell is a mixed population of CD4+ and CD8+ T cells.
• aspects of the disclosure are further drawn towards a nucleic acid construct encoding a chimeric antigen receptor.
• the chimeric antigen receptor comprises an extracellular ligand binding domain that is specific for a first antigen and a second antigen on the surface of a cancer cell, wherein the first antigen comprises CAIX and the second antigen comprises CD70.
• the nucleic acid construct further encodes for a transmembrane polypeptide, an intracellular signaling domain, and/or a co-stimulatory domain.
• the nucleic acid construct further encodes for a recombinant polypeptide.
• Aspects of the disclosure are further drawn towards a vector comprising the nucleic acid construct described herein.
• aspects of the disclosure are drawn towards a cell comprising the vector described herein.
• aspects of the disclosure are also drawn towards a method for treating a subject afflicted with cancer.
• the method comprises administering to a subject a therapeutically effective amount of the engineered cell described herein.
• aspects of the disclosure are drawn towards a method of reducing progression or promoting regression of a cancer in a subject.
• the method comprises administering the subject a therapeutically effective amount of the engineered cell described herein.
• aspects of the disclosure are drawn towards a method of reducing cellular proliferation of a cancer cell in a subject.
• the method comprises administering the subject a therapeutically effective amount of the engineered cell described herein.
• the cancer comprises renal cell carcinoma.
• a chimeric antigen receptor comprising an extracellular ligand binding domain, wherein the extracellular ligand binding domain is specific for a first antigen and a second antigen on the surface of a cancer cell, wherein the first antigen comprises CAIX and the second antigen comprises CD70.
• the CAR further comprises a transmembrane polypeptide, an intracellular signaling domain, and/or a co-stimulatory domain.
• the extracellular ligand binding domain comprises an antibody or fragment thereof.
• aspects of the invention are drawn towards a cell comprising the chimeric antigen receptor (CAR) as described herein.
• aspects of the invention are drawn towards an engineered cell comprising a first chimeric antigen receptor and a second chimeric antigen receptor, wherein the first chimeric antigen receptor comprises an extracellular ligand binding domain specific for CAIX, and wherein the second chimeric antigen receptor comprises an extracellular ligand binding domain that is specific for CD70.
• the engineered cell expresses and secretes a recombinant polypeptide.
• the first chimeric antigen receptor and the second chimeric antigen receptor are expressed from a single nucleic acid construct.
• FIG. 1 shows a schematic of bispecific tandem CAR T anti-CD70 and anti-CAIX scFvs were combined in different permutations by changing the order of the two targeting scFvs with various linkers and hinges connected to a costimulatory domain (non-limiting examples comprising CD28, 41BB, CD28-41BB, or 41BB-CD28) and an activating domain (such as, CD3).
• a costimulatory domain non-limiting examples comprising CD28, 41BB, CD28-41BB, or 41BB-CD28
• an activating domain such as, CD3
• the 2nd generation CAR T cell factories can be designed with the introduction of the second cassette anti-CD70 scFv.
• FIG. 2 shows that 2nd generation CAR T cell factories can be established by combining a series of CARs (such as different scFvs, linkers, and hinges) with immune checkpoint blockade as payload. Based on these data, 8 constructs were established by using G36 as the anti-CAIX scFv and B7 as the anti-CD70 scFv. The skilled artisan will recognize that any scFvs can be utilized according to the invention. The dual CAR engineering is the most important part and the payload was replaced with zsgreen to indicate the transduction efficiency.
• CARs such as different scFvs, linkers, and hinges
• FIG. 3 shows CAIX and CD70 upregulated and co-expressed on ccRCC.
• IHC staining of ccRCC primary cell lines generated from patient samples indicates that CAIX and CD70 are both highly expressed and co-expressed on ccRCC.
• CAIX and CD70 are two potential targets for ccRCC therapy.
• To validate targets we conducted IHC for staining of CAIX and CD70 in primary cell line which is generated from ccRCC patients. From these images, we can see that CAIX and CD70 staining are 100% positive, demonstrating that CAIX and CD70 are highly and simultaneously expressed in ccRCC. This is presently being validated in a study which determines CAIX and CD70 expression on ccRCC patients with at least 150 samples.
• FIG. 4 shows CAIX and CD70 upregulated and co-expressed on ccRCC. IHC staining of ccRCC patient samples indicates that CAIX and CD70 are both highly expressed and co-expressed on ccRCC.
• FIG. 5 shows establishment of CRISPR skrc-59 cell lines.
• four CRISPR engineered skrc-59 cell lines were established with four different phenotypes, (i) CAIX+ CD70+, (ii) CAIX+ CD70-, (iii) CAIX- CD70+, and (iv) CAIX- CD70-.
• Four CRISPR skrc-59 cell lines have been established.
• Four different phenotypes, (i) CAIX+ CD70+, (ii) CAIX+ CD70-, (iii) CAIX- CD70+, and (iv) CAIX- CD70- are used for the in vitro assay described herein.
• the corresponding table shows CAIX and CD70 expression level on 4 different cell lines that were quantified.
• FIG. 6 shows graphs of anti-CD70 minibodies that showed selective binding to CD70+ SKRC59 cells.
• Phage display (panned against CD70+ skrc-59 cells and subtracted against CD70- skrc-59 cells) indicates that a series of anti-CD70 minibodies showed binding with CD70 positive ccRCC skrc-59 cells.
• Anti-CD70 minibodies were expressed via Expi293 cells in a 6 well plate. 3 days after transfection, the supernatant was harvested and IgG quantification ELISA (Bethyl) was conducted to approximate the concentration of minibody in the supernatant. This approximate concentration was used to normalize the supernatants for FACS binding curves.
• FIG. 7 demonstrates anti-CD70 CAR T cells show killing activity in Celigo killing assay.
• These anti-CD70 scFv candidates were cloned into vectors and were packaged into lentiviruses to transduce primary T cells.
• CAR T cells were assessed for antiproliferation activity in Celigo assay. Results showed that #3, #7, #9 have efficacy compared to CD70 ligand CD27 CAR T cells.
• these hits were cloned into pHAGE vector and conducted Celigo killing assay. These graphs showed that anti-CD70 CAR T cells exhibited killing activity in Celigo killing assay with Effector: Target ratio of 2: 1.
• FIG. 8 demonstrates anti-CD70 CAR T cells show killing activity in Chromium 51 killing assay on CAIX+ CD70+ cells. CAR T cells were also assessed for killing activity in Chromium 51 release assay. Results showed that #7 CAR T cells have the enhanced efficacy compared to CD27 (CD70 ligand) CAR T cells.
• a chromium 51 4h release assay was conducted. After 4h incubation with chromium 51 labeled target cells, it validated that B7 has killing activity against SKRC59 CD70+ cells.
• FIG. 9 shows 2nd generation CAR-T cells.
• CAR T cells can be generated by using zsGreen instead of immune checkpoint blockade. Based on these data, 8 constructs were established by using G36 as the anti-CAIX scFv and B7 as the anti-CD70 scFv. The dual CAR engineering is the most important part and the payload was replaced with zsgreen to indicate the transduction efficiency.
• FIG. 10 shows transfected 293T cells binding with CAIX-PE.
• 293T cells were transfected with different constructs of dual CAR and the binding assay with PE labeled CAIX protein was performed. All dual CARs bind to CAIX, and different orientation of two scFvs influence the EC50 of anti-CAIX scFv binding.
• Anti-CAIX scFv G36 prefer the 2nd cassette after linker.
• 293T cells were transfected with these 8 bispecific constructs and also corresponding monoCARs and stained with CAIX-PE. After
• Anti-CAIX scFv G36 prefer to in the 2nd cassette after linker.
• FIG. 11 shows transfected 293T cells binding with CD70-APC.
• 293T cells were transfected with different constructs of dual CAR and the binding assay was performed with PE labeled CAIX protein. All dual CARs bind to CD70, and different orientation of two scFvs does not influence the EC50 of anti-CD70 scFv binding. Anti-CD70 scFv B7 does not have a significant preference.
• 293T cells were transfected with these 8 bispecific constructs and corresponding monoCARs and stained with CD70-PE. After normalization by transfect efficiency, different orientation of the two scFvs does not influence the EC50 of anti-CD70 scFv binding.
• FIG. 12 shows B7-GGGGS3-G36 killing assay with Celigo.
• B7-GGGGS3-G36 CAR for example was taken and selective killing assay was performed on mixed CAIX+ CD70+ dual positive cells with CAIX+ or CD70+ single positive or CAIX- CD70- cells.
• B7- GGGGS3-G36 CAR T cells had more killing activity on the targeted cells (CAIX+ CD70+) than non-targeted cells (CAIX+ CD70-, CAIX- CD70+, CAIX- CD70-).
• FIG. 13 shows B7-GGGGS3-G36 killing assay with FACS.
• Four different CRISPR engineered skrc-59 cells were transduced with BFP fluorescence group.
• Cells were mixed with 1 : 1 : 1 : 1 ratio and were treated with B7-GGGGS3-G36 CAR T cells or culture medium.
• stain cells with PE labeled anti-CD70 antibody and APC labeled anti-CAIX antibody and, perform flow cytometry. It showed that B7-GGGGS3-G36 has selective killing to CAIX+ CD70+ cells whose population is reduced from 26.7% to 18.5%.
• FIG. 14 is a schematic of fine-tuned anti-CAIX CAR T.
• anti-CAIX scFvs with various KDs were generated as CARs and corresponding killing activity was assessed.
• the 2 nd generation CAR T cell factories have been designed with the introduction of the second cassette anti-CD70 scFv.
• FIG. 15 is a diagram rendering of fine-tuned anti-CAIX CAR-T cells.
• CAR T cells were generated by using zsGreen instead of immune checkpoint blockade.
• 8 constructs were established by using G36 as the anti-CAIX scFv and B7 as the anti-CD70 scFv.
• the dual CAR engineering is an important part and the payload was replaced with zsgreen to indicate the transduction efficiency.
• a series of antibodies against CAIX were identified with different KD values from 1.49nM to 99.58nM (See also FIG. 16). 13 antibodies were classified into 4 groups by binding experiment.
• FIG. 16 depicts table showing 19 anti-CAIX ScFvs identified and the corresponding binding.
• FIG. 17 shows graphs of anti-CAIX monoCAR T killing on CAIX+CD70+. These anti-CAIX CAR T cells were assessed for antiproliferation activity in Celigo assay. Results showed based on killing activity, 19 CARs can be separated into 4 groups. G37, G39, G125 (++++) > G10, G21, G36, G40, G45, G57, G62, G98, G106, G119 (+++) > G6, G9, G17, G27, G28 (++) > G104 (++).
• FIG. 18 shows graphs of Anti-CAIX monoCAR T killing on CAIX+CD70+. These anti-CAIX CAR T cells were assessed for antiproliferation activity in Celigo assay. Results showed based on killing activity, thatl9 CARs can be separated into 4 groups.
• FIG. 19 shows Anti-CAIX monoCAR T killing on CAIX+CD70+. These anti-CAIX CAR T cells were assessed for antiproliferation activity in Celigo assay. Results showed based on killing activity, 19 CARs can be separated into 4 groups. G37, G39, G125 (++++) > G10, G21, G36, G40, G45, G57, G62, G98, G106, G119 (+++) > G6, G9, G17, G27, G28 (++) > G104 (++).
• FIG. 20 is a schematic of a selective killing assay (e.g., using either a Celigo assay or FACS assay).
• a selective killing assay e.g., using either a Celigo assay or FACS assay.
• 2 different selectivity assay were performed. For example, CAIX+ CD70+ cells were mixed with mono positive cells and then CART cells were added. After certain time incubation, the number of double positive cells and single positive cells were measured through Celigo.
• FIG. 24 shows CAIX and CD70 upregulated and co-expressed on ccRCC.
• FIG. 25 shows design of CARs to address unwanted side-effects previously associated with CART cell therapy.
• CART cell therapy we elevate the efficacy and safety profiling.
• CAR-T cell factories were developed, which is able to modulate the tumor microenvironment through secreting human anti-immune checkpoint inhibitor monoclonal antibodies (mAbs) locally at the tumor site to reverse T cell exhaustion.
• mAbs human anti-immune checkpoint inhibitor monoclonal antibodies
• fine-tuned CARs were designed with reduced affinity scFv to expand the therapeutic window by limiting the recognition of the tumor associated antigen on normal tissues.
• FIG. 26 shows CAR-T cell factories show enhanced killing.
• the CAR-T cell factories are able to target CAIX and secrete checkpoint blockade inhibitor at the tumor site to convert suppressive tumor microenvironment.
• 1E7 CAR-T cells were injected by i.v. on Day 0 and 2.5E6 CAR-T cells were injected on Day 17.
• CAR-T cell factories showed enhanced killing efficacy compared to the CAR-T cells secreting irrelevant antibodies in our orthotopic RCC mouse model.
• FIG. 27 shows schematic of assessment of CAR-T on humanized orthotopic ccRCC mouse model.
• FIG. 28 shows MRI imaging of RCC mouse model.
• FIG. 29 shows a schematic of an experiment designed to establish stress model and compare 2nd generation and 3rd generation CAR and CD8 vs. CD4/8 on orthotopic ccRCC mouse model.
• FIG. 30 shows 4 IBB performed superior killing in vivo. Efficient tumor regression was observed in G36-41BB and G36-CD28-41BB treated group at the dose of 1E7. Mixed CD4/CD8 G36-41BB CAR-T performed superior killing even at the dose of 3E6. Comparing two 2 nd generation CAR constructs and one 3 rd generation CAR construct, G36-41BB outperformed than G36-CD28 and G36-CD28-41BB
• FIG. 31 shows G36-41BB stress model. Dose of 3E6 can be used for G36-41BB stress model.
• FIG. 32 shows 4 IBB performed superior killing in vivo.
• FIG. 33 shows 4 IBB CAR-T cell performed the best expansion in vivo.
• FIG. 34 shows CD 8 vs. CD4/CD8.
• FIG. 35 shows CD4 and CD8 T cells expansion in vivo.
• FIG. 36 shows a schematic of a fine-tuned anti-CAIX CAR-T which has been designed to limit on-target off-tumor effect. Without wishing to be bound by theory, lowering the affinity of anti-CAIX scFv, the CAR only recognizes the high density of CAIX on ccRCC. A series of anti-CAIX scFvs with various KDs were cloned into lentiviral vectors and corresponding CAR-T cells were generated and assessed the killing activity.
• FIG. 37 is a schematic detailing the "therapeutic window”.“Therapeutic window” is a term originally from pharmaceutical toxicology and can refer to a range of doses between efficacy and toxicity, achieving the highest therapeutic benefit without resulting in unacceptable toxicity; it is the range between the minimum effective dose (MED) and the maximum tolerated dose (MTD. This concept was applied to optimize CAR-T therapy. To expand the therapeutic window, we can fine tune the CAR affinity with the antigens by assembling the scFvs with different KDs in the CAR construct, such as from lnm to lOOnm. Ideally, after optimization, the CAR only recognize the high density antigens on tumor cells not the low density antigens on normal cells.
• MED minimum effective dose
• MTD maximum tolerated dose
• Targeting antigens expressed exclusively on tumor cells or antigens that are expressed only on non-critical tissues widens the therapeutic window as direct toxicity on vital tissues would not occur.
• targeting antigens that are expressed in critical normal tissues/cells narrows the window by decreasing MTD
• FIG. 38 is a schematic describing expanding the therapeutic window to address on- target off-tumor side effects.
• FIG. 39 shows IHC double staining of CAIX and CD70 on patient samples.
• CD70 and CAIX are highly expressed and co-expressed on ccRCC.
• CD70 we choose CD70 as our second target.
• FIG. 40 shows IHC double staining of CAIX and CD70 on patient samples.
• FIG. 41 shows a panel of scFvs against CAIX with different KD values from 1.49nM to 99.58nM as the table shows.
• Corresponding CAR-T cells were generated and screened by Celigo killing assay
• FIG. 42 shows that there is correlation between affinity of scFv and killing of CART cells, 19 CARs were divided into 4 groups according to the cytotoxicity and to be tested against skrc-59 cells with different expression levels of CAIX.
• FIG. 43 shows "Or” gating to capture tumor cell heterogeneity but not kill low target density healthy cells.
• FIG. 44 shows CD70 can be an ideal target as the second target for our bispecific CAR since it’s highly expressed on kidney cancer, especially clear cell renal cell carcinoma.
• FIG. 45 shows IHC double staining of CAIX and CD70 on ccRCC patient samples.
• CD70 and CAIX are highly expressed and co-expressed on ccRCC.
• CD70 we choose CD70 as our second target.
• FIG. 46 shows IHC double staining of CAIX and CD70 on ccRCC patient samples.
• CD70 and CAIX are highly expressed and co-expressed on ccRCC.
• CD70 we choose CD70 as our second target.
• FIG. 47 shows CAIX and CD70 upregulated and co-expressed on ccRCC.
• FIG. 48 shows anti-CD70 minibodies showed selective binding to CD70+ SKRC59 cells.
• phage display panned against CD70+ skrc-59 cells and subtracted against CD70- skrc-59 cells
• Anti-CD70 minibodies were expressed via Expi293 cells in a 6 well plate. 3 days after transfection, the supernatant was harvested and IgG quantification ELISA (Bethyl) was conducted to approximate the concentration of minibody in the supernatant. This approximate concentration was used to normalize the supernatants for FACS binding curves.
• FIG. 49 shows anti-CD70 B7 CAR-T cells showed promising killing against CD70+ skrc-59 cells.
• phage display panned against CD70+ skrc-59 cells and subtracted against CD70- skrc-59 cells
• we discovered a series of anti-CD70 minibodies which showed promising binding with CD70 positive ccRCC skrc-59 cells.
• these hits were cloned into lentiviral vector and corresponding CART cells were conducted Celigo killing assay. From the screening, we found B7 as the candidate.
• FIG. 50 shows bispecific CAR constructs.
• a series of constructs were established by using G36 as the anti-CAIX scFv and B7 as the anti-CD70 scFv in different rotations (or orientations) with different linkers.
• FIG. 51 shows that anti-scFv G36 prefer to be in the 2nd cassette after the linker.
• Anti-CAIX scFv G36 prefer to in the 2 nd cassette after linker.
• FIG. 52 shows that anti-CD70 scFv B7 has no preference. Transfect 293T cells with different constructs of dual CAR and perform the binding assay with APC labeled CD70 protein. After normalization by transfection efficiency, it shows that different orientation of two scFvs doesn't influence the EC50 of anti-CD70 scFv binding. Anti-CD70 scFv B7 doesn’t have a significant preference.
• FIG. 53 shows that establishment of 4 CRISPR skrc-59 cell lines.
• FIG. 54 shows B7-GGGGS3-G36 shows preferential killing.
• B7-GGGGS3-G36 CAR Take B7-GGGGS3-G36 CAR as example we performed selective killing assay on mixed CAIX+ CD70+ dual positive cells, with CAIX+ or CD70+ single positive cells, or CAIX- CD70- cells. It showed that B7- GGGGS3-G36 CAR-T cells have preferential killing activity against the targeted cells (CAIX+ CD70+) than non-targeted cells (CAIX+ CD70-, CAIX- CD70+, CAIX- CD70-). After certain time incubation, the number of double positive cells and single positive cells were measured through Celigo. And we can see that B7-GGGGS3 has limited selective index on CAIX+CD70+ while mixed with CAIX+ cells.
• FIG. 55 shows that B7-GGGGS3-G36 shows preferential killing.
• 4 different CRISPR engineered skrc-59 cells were transduced with BFP fluorescence group.
• Cells were mixed with 1 : 1 : 1 : 1 ratio and were treated with B7-GGGGS3-G36 CAR-T cells or culture medium.
• stain cells with PE labeled anti-CD70 antibody and APC labeled anti-CAIX antibody and, perform flow cytometry. It showed that B7-GGGGS3-G36 has selective killing to CAIX+ CD70+ cells whose population is reduced from 26.7% to 18.5%.
• In this selective killing data from FACS it showed that B7-GGGGS3-G36 has selective killing to CAIX+ CD70+ cells whose population is reduced from 26.7% to 18.5%.
• FIG. 56 shows a schematic of a bispecific split CAR T.
• Anti-CD70 and anti-CAIX scFvs were expressed on cell surface with different costimulation domains. See, for example, dual(split) CAR T, Nat Rev Cancer 16(9): 566-81.
• FIG. 57 shows split CAR killing on ccRCC primary cancer cell.
• Split CAR T cells were assessed killing activity on primary ccRCC cancer cells with monoCAR T and Tandem CAR T cells. It showed that split CAR achieved a superior killing at low E:T ratio, like 1 : 1 ratio.
• FIG. 58 provides multiple sequence alignment of amino acid sequences of anti- carbonic anhydrase IX (G250) scFv clones.
• FIG. 59 provides alignment of human and mouse CAIX amino acid sequences.
• FIG. 60 provides structure of carbonic anhydrase IX (G250).
• FIG. 61 shows CAR T cell killing assay of Skrc-59 transduced cells. Celigo image cytometry is shown.
• FIG. 62 shows homology study of 10 different species. Tree: distance on each branch equals number of differences between sequences (e.g. 0.1 means 10 % differences between two sequences); distance between 2 species equals sum length of all branches connecting them homology > 60% - potential cross reactivity.
• FIG. 63 shows establishment of skrc-59 stable cell lines expressing CAIX or CD70 from different species.
• CRISPR knock out CAIX-/CD70- skrc-59 cells - transduced with the 10 different constructs (CD70 or CAIX from the 5 different species) - sorted by FACS.
• FIG. 64 shows binding assay of anti-CAIX(G36) scFv. Normalized with the binding data of commercial antibodies. Analyzed using a non-linear regression and a log (agonist) vs. response model.
• FIG. 65 shows killing assay of anti-CAIX (G36) CAR T cells. Monkey 100% (same killing as human, cross reactive); mouse, hamster 50% (significant killing).
• FIG. 66 shows killing assay of anti-CD70 (B7) CAR T cells.
• FIG. 67 shows killing assay of 20 different anti-CAIX CAR T cells.
• FIG. 68 shows fold change killing assay G36 E:T 10: 1.
• FIG. 69 shows killing assay G36 E:T 5: 1.
• FIG. 71 shows killing assay 20 anti-CAIX scFvs.
• FIG. 72 shows all candidates monkey.
• FIG. 73 shows all candidates mouse.
• FIG. 74 shows all candidates hamster.
• FIG. 75 shows fold change B7 killing assay.
• FIG. 76 shows the amino acid sequence and germline alignment of anti-cd70 antibodies.
• FIG. 77 shows a nucleic acid construct of a split car.
• FIG. 78 shows the amino acid sequence and germline alignment of anti-PDLl and anti -PD 1 sequences.
• FIG. 79 shows the amino acid sequence and germline alignment of anti-CAIX antibodies.
• FIG. 80 shows tandem CAR B7-GGGGS5-G36 cytotoxicity.
• FIG. 81 shows the amino acid sequence and germline alignment of anti -TI GIT antibodies.
• FIG. 82 shows the anti-PD-Ll amino acid sequences.
• FIG. 83 shows the anti-PDl nucleic acid and amino acid sequences.
• Chimeric Antigen Receptor (CAR) T-cell therapies represent an exciting area of discovery that has already revolutionized treatment in several blood-borne cancers.
• ALL Acute Lymphoblastic Leukemia
• remission rates were demonstrated to be 80 to 90 percent, leading to FDA approval.
• the technology can utilize a patient’s own immune cells to fight their cancer by engineering them to better recognize particular proteins located on the cancer cells. For example, after that change has been effected in the lab, the immune cells are grown extensively in the lab, multiplying their cancer-killing potential, and finally infused back to the patient where they can attack cancer wherever it is in the body with increased efficacy and numbers.
• CAR T-cell therapies have been largely elusive in solid tumors such as renal cell carcinoma (RCC) because the cancer cells create a tumor microenvironment that turns off immune penetration.
• RCC renal cell carcinoma
• Our approach relies upon the creation of humanized RCC in mice that can be studied to better understand their immunologic fingerprint. Examining how human RCC behaves in mice has allowed the lab to identify antigens, such as Carbonic Anhydrase IX [CAIX] and CD70, that could be targeted using CAR T-cell therapy in humans and other mammals. To large degree, these two antigens, namely CAIX and CD70, are only found together on kidney cancer tumor cells, allowing the immune system to attack locally with minimal impact on healthy tissue. There have also been advances in counteracting the immunosuppressive effects within the microtumor environment by engineering T-cells that produce and/or secrete check-point inhibitor antibodies. Pairing these approaches could allow for dramatically improved efficacy within the tumor microenvironment.
• CAR T-cells that target CAIX and CD70, for example, that can be administered to animals with close monitoring for changes in the tumor
• Chimeric-antigen receptor (CAR) T-cell therapies redirect a patient’s T-cells to kill tumor cells by the exogenous expression of a CAR.
• a CAR is a membrane spanning fusion protein that links the antigen recognition domain of an antibody or fragment to the intracellular signaling domains of the T-cell receptor and co receptor.
• chimeric antigen receptors fuse antigen-specific antibody fragments to T-cell co-stimulatory domains and the CD3 zeta intracellular signaling domain, allowing for the re-direction of T-cells towards an antigen presented on a cell of interest, for example, onto tumor cells.
• antibody herein is used in the broadest sense and can refer to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds
• Antibodies of the disclosure can include, but are not limited to, polyclonal, monoclonal, humanized, fully human, bispecific, multispecific, chimeric, dAb (domain antibody), single chain antibodies, Fab, Fab' and F(ab')2 fragments, scFvs, diabodies, minibodies, scFv-Fc fusions, and Fab expression libraries.
• any reference to“antibody” or“antibodies” made herein encompasses, for example, any (or all) of these molecules so long as they exhibit the desired antigen-binding activity.
• a single chain Fv (“scFv”) polypeptide molecule is a covalently linked VH::VL heterodimer, which can be expressed from a gene fusion including VH- and VL-encoding genes linked by a peptide-encoding linker.
• scFv single chain Fv
• a number of methods have been described to discern chemical structures for converting the naturally aggregated, but chemically separated, light and heavy polypeptide chains from an antibody V region into an scFv molecule, which will fold into a three dimensional structure substantially similar to the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos. 5,091,513; 5,132,405; and 4,946,778.
• TME tumor microenvironment
• B7-GGGGS-G36 CAR T cells had more killing activity on the targeted renal carcinoma cells (CAIX+ CD70+) than non-targeted cells (CAIX+ CD70-, CAIX- CD70+, CAIX- CD70-).
• the bispecific CAR can also (b) secrete a checkpoint blockade antibody that removes repression in the tumor microenvironment.
• Other embodiments comprise a fine- tuned CAR that only recognizes the high density antigens on tumor cells, not the low density antigens on normal cells. Without wishing to be bound by theory, this can be accomplished by, for example, lowering the affinity of the antibody or antibodies associated with the CAR.
• FIG 1, for example, provides a schematic of a bispecific CAR that targets two antigens, such as CAIX and CD70.
• Bispecific CARs can refer to CARs that has binding specificities for at least two different antigens.
• bispecific CARs can comprise monoclonal antibodies, such as human or humanized antibodies, or fragments thereof.
• one of the binding specificities is CAIX and/or CD70.
• the second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.
• one of the binding specificities is for CAIX and the second binding specificity is for CD70.
• Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab')2 bispecific antibodies). Methods for making bispecific antibodies are known in the art. See for example U.S. patent 8,329,178, which is incorporated herein by reference in its entirety.
• Antibody molecules obtained from humans relate to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgGl, IgG2, and others.
• the light chain may be a kappa chain or a lambda chain.
• the term“antigen-binding site,” or“binding portion” can refer to the part of the immunoglobulin molecule that participates in antigen binding.
• the antigen binding site is formed by amino acid residues of the N-terminal variable (“V”) regions of the heavy (“H”) and light (“L”) chains.
• V N-terminal variable
• L light
• FR refers to amino acid sequences which are naturally found between, and adjacent to, hypervariable regions in immunoglobulins.
• the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen-binding surface.
• the antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as
• CDRs complementarity-determining regions
• An emerging mechanism associated with the progression of tumors is the immune checkpoint pathway, which include cellular interactions that prevent excessive activation of T cells under normal conditions, allowing T cell function in a self-limited manner.
• immune checkpoint pathway As an evasion mechanism, many tumors are able to stimulate the expression of immune checkpoint molecules, resulting in an anergic phenotype of T cells that cannot restrain tumor progression.
• emerging clinical data highlight the importance of one inhibitory ligand and receptor pair as an immune checkpoint: the programmed death-ligand 1 (PD-L1; B7-H1 and CD274) and programmed death receptor-1 (PD-1; CD279), in preventing killing of cancer cells by cytotoxic T-lymphocytes.
• PD1 receptor is expressed by many cell types like T cells, B cells, Natural Killer cells (NK) and host tissues. Tumors and Antigen- presenting cells (APC) expressing PD-L1 can block T cell receptor (TCR) signaling of cytotoxic T-lymphocytes through binding to receptor PD-1, decreasing the production of cytokines and T cell proliferation.
• TCR T cell receptor
• PD-L1 overexpression can be found in many tumor types and may also mediate an immunosuppressive function through its interaction with other proteins, including CD80 (B7.1), blocking its ability to activate T cells through binding to CD28.
• CAR tumor-directed chimeric antigen receptors
• CARTS lymphocytes
• the cells comprise a split CAR, such as anti-CD70 and anti- CAIX scFvs expressed on cell surface with different costimulation domains.
• some embodiments comprise a fine-tuned CAR.
• the CARTS are further modified to express and secrete one or more polypeptides, such as for example an antibody or a cytokine, such as IL-12, IL-15, or IL-18.
• polypeptides such as for example an antibody or a cytokine, such as IL-12, IL-15, or IL-18.
• armed CARTS or CAR factories Such CARTS allow for simultaneous secretion of the polypeptide locally at the targeted site (i.e., tumor site).
• a split CAR comprises two or more CARs on the surface of a cell, such as a T cell or NK cell.
• the CARs can be specific for two or more antigens, such as CD70 and CAIX.
• FIG. 77 provides an example of a nucleic acid construct encoding split CARs.
• the first CAR is specific for CAIX
• the second CAR is specific for CD70.
• the CARs can be in any orientation desired.
• the first CAR can be specific for CD70 and the second CAR can be specific for CAIX.
• first and the second CARs can be expressed from a single nucleic acid construct.
• a nucleic acid encoding a cleavable linker can be located between the nucleic acids encoding the first and the second CAR.
• the two CARs can be expressed in the same cell but from two separate nucleic acid constructs.
• CAR chimeric antigen receptor
• the scFv presented in the CAR is linked to an intracellular signaling block that includes CD3z to induce T cell activation followed by antigen binding.
• This structure is characteristic for first- generation CARs, which were improved to second- and generation CARs that link the signaling co-stimulatory endodomains of CD28, 4-1BB, or 0X40 to CD3 or 3rd-generation CARs that links two elements to CD3z in tandem.
• CART cells antigen-presenting cells
• the lymphocytes can include a receptor that is chimeric, non natural and engineered at least in part by the hand of man.
• the engineered chimeric antigen receptor has one, two, three, four, or more components, and in some embodiments the one or more components facilitate targeting or binding of the lymphocyte to one or more tumor antigen-comprising cancer cells.
• the CAR according to the disclosure comprises at least one transmembrane polypeptide comprising at least one extracellular ligand-biding domain and; one
• transmembrane polypeptide comprising at least one intracellular signaling domain; such that the polypeptides assemble together to form a Chimeric Antigen Receptor.
• Exemplary CARS useful in aspects of the disclosure include those disclosed in for example
• extracellular ligand-binding domain can refer to an oligo- or polypeptide that can bind a ligand.
• the domain can interact with a cell surface molecule.
• the extracellular ligand-binding domain can be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state.
• the extracellular ligand-binding domain can comprise an antigen binding domain or antigen recognition domain derived from an antibody against an antigen of the target.
• the antigen binding domain or antigen recognition domain can be an antibody fragment.
• An "antibody fragment” can be a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments. Referring to FIG. 1, for example, one embodiment comprises a CAR with two scFvs as the antigen recognition domains.
• one embodiment comprises a CAR with one scFv as the antigen recognition domain.
• the antigen recognition domain can be directed towards any antigen target of interest.
• the antigen target of interest is on the surface of a cell, such as the surface of a cancer cell.
• Non-limiting examples of antigen targets comprise CAIX and/or CD70.
• the CAR is specific for CAIX and/or CD70.
• said extracellular ligand-binding domain is a single chain antibody fragment (scFv) comprising the light (VL) and the heavy (VH) variable fragment of a target antigen specific monoclonal antibody joined by a flexible linker.
• scFv single chain antibody fragment
• VL light
• VH heavy
• embodiments can comprise different linkers which are typically known in the art. See, for example, Chen, et al. "Fusion protein linkers: property, design and functionality. "Advanced drug delivery reviews 65.10 (2013): 1357-1369, which is incorporated by reference herein in its entirety. For example, using different linkers will allow one to fine tune the dual-targeting CAR construct.
• the linker length can vary depending on the antibodies of the dual-targeting CAR construct, their angle of approach to the target epitope, topography of the target on the tumor cell membrane.
• the flexible linkers can include GGGS1, GGGGS3, GGGGS5, or IgGl hinge.
• the number of Gs in the linker can be 2, 3, 4, 5, 6, or 7 in combination with either SI, S2, S3, S4, S5, or S6.
• the scFv antibody is specific for CAIX and/or CD70.
• the orientations of the scFvs to the linker can vary.
• the anti-CAIX scFv can be in the first cassette (i.e., before the linker), and the anti-CD70 cassette can be in the second cassette (i.e., after the linker).
• the anti-CAIX scFv can be in the second cassette, and the anti-CD70 scFv can be in the first cassette.
• anti-CAIX scFv G36 can be in the first cassette, and anti-CD70 B7 can be in the second cassette.
• anti-CAIX scFv G36 can be in the second cassette, and anti- CD70 B7 can be in the first cassette.
• Linkers of various lengths and flexibilities can be utilized as described herein. As shown, different orientations of the two scFvs can influence binding. For example, G36 has higher binding what engineered as the second cassette. On the other hand, as shown in FIG. 11, anti-CD70 scFv B7 does not have significant preference.
• Examples of antibodies useful in constructing the CARs according to the disclosure include those detailed in Table 1, 2, 3 or 4 herein. See also, for example, WO/2007/065027 and WO/2017/100985, the contents of which are hereby incorporated by reference in their entireties.
• the antigen recognition domain useful in constructing the CAR-Ts can be synthesized, engineered, and/or produced using nucleic acids (e.g., DNA).
• the DNA encoding the antigen recognition domain can be cloned in frame to DNA encoding necessary CAR-T elements such as, but not limited to, CD8 hinge regions, transmembrane domains, co-stimulatory domains of molecules of immunological interest such as, but not limited to, CD28 and 41BB and CD3-zeta intracellular signaling domains. See FIG. 2, for example.
• Binding domains other than scFv can also be used for predefined targeting of lymphocytes, such as camelid single-domain antibody fragments or receptor ligands, antibody binding domains, antibody hypervariable loops or CDRs as non-limiting examples.
• the transmembrane domain further comprises a stalk region between said extracellular ligand-binding domain and said transmembrane domain.
• the term "stalk region" used herein can mean any oligo- or polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain.
• stalk region are used to provide more flexibility and accessibility for the extracellular ligand-binding domain.
• a stalk region may comprise up to 300 amino acids, for example, 10 to 100 amino acids or, for example, 25 to 50 amino acids.
• Stalk region may be derived from all or part of naturally occurring molecules, such as from all or part of the extracellular region of CD8,
• the stalk region may be a synthetic sequence that corresponds to a naturally occurring stalk sequence, or may be an entirely synthetic stalk sequence.
• said stalk region is a part of human CD 8 alpha chain.
• the signal transducing domain or intracellular signaling domain of the CAR of the invention is responsible for intracellular signaling following the binding of
• the signal transducing domain is responsible for the activation of at least one of the normal effector functions of the immune cell in which the CAR is expressed.
• the effector function of a T cell can be a cytolytic activity or helper activity including the secretion of cytokines.
• the term "signal transducing domain" can refer to the portion of a protein which transduces the effector signal function signal and directs the cell to perform a specialized function.
• Signal transduction domain can comprise two distinct classes of cytoplasmic signaling sequence, those that initiate antigen-dependent primary activation, and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal.
• Primary cytoplasmic signaling sequence can comprise signaling motifs which are known as immunoreceptor tyrosine-based activation motifs of ITAMs.
• ITAMs are well defined signaling motifs found in the intracytoplasmic tail of a variety of receptors that serve as binding sites for syk/zap70 class tyrosine kinases.
• Examples of ITAM used in the invention can include as non-limiting examples those derived from TCR zeta, FcR gamma, FcR beta, FcR epsilon, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b and CD66d.
• the signaling transducing domain of the CAR can comprise the CD3 zeta signaling domain, or the intracytoplasmic domain of the Fc epsilon RI beta or gamma chains.
• the signaling is provided by CD3 zeta together with co stimulation provided by CD28 and a tumor necrosis factor receptor (TNFr), such as 4-1BB or 0X40), for example.
• TNFr tumor necrosis factor receptor
• the intracellular signaling domain of the CAR of the disclosure comprises a co-stimulatory signal molecule. In some embodiments the intracellular signaling domain contains 2, 3, 4 or more co-stimulatory molecules in tandem.
• a co-stimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient immune response.
• Co-stimulatory ligand can refer to a molecule on an antigen presenting cell that specifically binds a cognate co-stimulatory molecule on a T-cell, thereby providing a signal which, in addition to the primary signal provided by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, mediates a T cell response, including, but not limited to, proliferation activation, differentiation and the like.
• a co stimulatory ligand can include but is not limited to CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, an agonist or antibody that binds Toll ligand receptor and a ligand that specifically binds with B7-H3.
• a co-stimulatory ligand also encompasses, inter alia, an antibody that specifically binds with a co-stimulatory molecule present on a T cell, such as but not limited to, CD27, CD28, 4-IBB, 0X40, CD30, CD40, PD- 1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83.
• an antibody that specifically binds with a co-stimulatory molecule present on a T cell such as but not limited to, CD27, CD28, 4-IBB, 0X40, CD30, CD40, PD- 1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83.
• a "co-stimulatory molecule” can refer to the cognate binding partner on a T- cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the cell, such as, but not limited to proliferation.
• Co-stimulatory molecules include, but are not limited to an MHC class 1 molecule, BTLA and Toll ligand receptor.
• costimulatory molecules examples include CD27, CD28, CD8, 4- IBB (CD 137), 0X40, CD30, CD40, 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.
• said signal transducing domain is a TNFR-associated Factor 2 (TRAF2) binding motifs, intracytoplasmic tail of costimulatory TNFR member family.
• Cytoplasmic tail of costimulatory TNFR family member contains TRAF2 binding motifs consisting of the major conserved motif (P/S/A)X(Q/E)E) or the minor motif (PXQXXD), wherein X is any amino acid.
• TRAF proteins are recruited to the intracellular tails of many TNFRs in response to receptor trimerization.
• Chimeric antigen receptors fuse antigen-recognition domains to signaling domains (also referred to as stimulatory domains) that modulate (i.e., stimulate) cell signaling.
• signaling domains also referred to as stimulatory domains
• Non-limiting examples of such stimulatory domains comprise those of CD28, 41BB, and/or CD3-zeta intracellular signaling domains. See FIG. 2, for example.
• transmembrane polypeptides comprise the ability to be expressed at the surface of an immune cell, in particular lymphocyte cells or Natural killer (NK) cells, and to interact together for directing cellular response of immune cell against a predefined target cell.
• the different transmembrane polypeptides of the CAR of the present invention comprising an extracellular ligand-biding domain and/or a signal transducing domain interact together to take part in signal transduction following the binding with a target ligand and induce an immune response.
• the transmembrane domain can be derived either from a natural or from a synthetic source.
• the transmembrane domain can be derived from any membrane-bound or transmembrane protein.
• amino acid sequence functional variants of the polypeptide can be prepared by mutations in the DNA which encodes the polypeptide.
• Such variants or functional variants include, for example, deletions from, or insertions or substitutions of, residues within the amino acid sequence. Any combination of deletion, insertion, and substitution may also be made to arrive at the final construct, provided that the final construct possesses the desired activity, especially to exhibit a specific anti-target cellular immune activity.
• the functionality of the CAR of the invention within a host cell is detectable in an assay suitable for demonstrating the signaling potential of said CAR upon binding of a particular target.
• this assay allows the detection of a signaling pathway, triggered upon binding of the target, such as an assay involving measurement of the increase of calcium ion release, intracellular tyrosine phosphorylation, inositol phosphate turnover, or interleukin (IL) 2, interferon .gamma., GM-CSF, IL-3, IL-4 production thus effected.
• IL interleukin
• a number of mAbs have been identified that react with surface antigens on RCC. These include mAbs that recognize differentiation and overexpressed antigens as well as mAbs that identify RCC-associated antigens not expressed in normal kidney (Michael, 2003; Yang, 2003).
• the gene for CAIX also known as G250 and MN is located on chromosomes 9pl2 to 13 and encodes a transmembrane protein that binds zinc and has CA activity (Zavada, 1997; Grabmaier, 2000).
• CAIX/G250/MN/ is found both at the plasma membrane and as a nuclear protein with apparent molecular weights of 58 and 54 kDa. It is N- glycosylated, and in the non-reduced state it forms oligomers (Pastorekova, 1992). Sequence analysis of the predicted CAIX protein shows that it contains a signal peptide (aa 1-37), an extracellular (EC) part (aa 38-414), a hydrophobic transmembrane region of 20 amino acids (aa 415-434) and a small C-terminus cytoplasmic portion of 25 amino acids (aa 435-459).
• the human and murine CAIX amino acid sequences are shown in Figure 59.
• the extracellular portion is composed of two distinct domains.
• the region between the signal peptide and the CA domain (aa 53-111) shows significant homology (38% identity) with a keratin sulfate attachment domain of a human large aggregating proteoglycan, aggrecan (Doege, 1991).
• a hexapeptide motif with consensus E-E-D- L-P-E SEQ ID NO: [ ]
• the carbonic anhydrase domain is located close to the plasma membrane (aa 135-391).
• the CAIX antigen appears at malignant
• Epitopes expressed on the cell surface of tumor cells are superior targets for humoral anti-cancer therapy since, unlike intracellular antigens, they are accessible to circulating antibodies in vivo.
• Human monoclonal antibodies (mAbs) have become a well- tolerated treatment option in an increasing number of cancers.
• the concept of selective tumor targeting with antibodies is based on the avid interaction between the antibody and an antigen that is expressed on malignant cells, but not on normal tissues.
• Many mechanisms have been proposed for the ability of antibodies against tumors to mediate their effects in vivo. For example, engagement of the antibody Fc domain with effector cell FcyRs leads to antibody-dependent cell-mediated cytotoxicity (ADCC).
• ADCC antibody-dependent cell-mediated cytotoxicity
• Some (antagonist or inhibitory) antibodies can block the signaling on tumor cells and in this way may act synergistically with immune effector responses by rendering the tumor cells more susceptible to immune effector cell triggered apoptosis or lytic cell death (Baselga, 1998).
• Another way that antibodies can be utilized is through the construction and functional expression of chimeric-immune receptors or“T-bodies” on T-lymphocytes otherwise known as“designer T-cells”.
• the antigen binding domain of the chimeric receptor can consist of an antigen-binding domain, for example, a single-chain antibody (scFv), while the intracellular signaling domain is derived from the cytoplasmic part of a membrane-bound receptor that can induce cellular activation (Maher, 2002; Pinthus, 2003).
• T-lymphocytes grafted with a chimeric receptor have the combined advantages of MHC-independence and antibody-based antigen binding with efficient T-cell activation upon specific binding to the receptor ligand. This activation results in the production and secretion of cytokines such as IL-2, interferon, GM-CSF and TNF-a.
• cytokines such as IL-2, interferon, GM-CSF and TNF-a.
• Antigen-specific lysis of tumor cells both in vitro and in vivo have been reported.
• T- lymphocytes can be permanently grafted with antigen-specific chimeric receptors by retroviral transduction of vector construct
• Embodiments of the present invention can comprise an isolated human monoclonal antibody or fragment thereof that immunospecifically binds to a carbonic anhydrase IX (G250) protein.
• Such antibodies can reduce carbonic anhydrase activity of said protein.
• anti-CAIX antibodies can include those described in WO
• embodiments of the invention comprise single chain antibodies, such as scFvs G6, G9, G10, G17, G21, G27, G28, G36, G37, G39, G40, G45, G57, G62, G98, G104, G119, or G125 as well as any other scFvs identified according to the methods disclosed herein.
• scFvs G6, G9, G10, G17, G21, G27, G28, G36, G37, G39, G40, G45, G57, G62, G98, G104, G119, or G125 as well as any other scFvs identified according to the methods disclosed herein.
• amino acids of the CDRs of the anti-CAIX antibodies are provided in Table 1:
• CAIX antibodies are provided in Table 2:
• Embodiments can also comprise a consensus sequence of any of the amino acid sequences described herein. For example, when four or more clones have the same amino acid at a given position, that position in the consensus is designated by that amino acid.
• CD70 is found on the surface of tumors cells of the kidney (clear cell carcinoma and papillary carcinoma, for example), pancreas, larynx or pharynx, melanoma, ovary, lung adenocarcinoma, colon, breast, and brain. See, for example, British Journal of Cancer 103 (2010) 676-684.
• Embodiments of the present invention can comprise an isolated human monoclonal antibody or fragment thereof that immunospecifically binds to CD70.
• embodiments of the invention comprise single chain antibodies, such as scFvs A20, B2, B3, B5, B7, B8, or B9 as well as any other scFvs identified according to the methods disclosed herein.
• amino acids of the CDRs of the anti-CD70 antibodies are provided in Table 3:
• amino acid sequences of the VH and VL regions of the anti- cd70 antibodies are provided in Table 4:
• Embodiments can also comprise a consensus sequence of any of the amino acid sequences described herein. For example, when four or more clones have the same amino acid at a given position, that position in the consensus is designated by that amino acid.
• Embodiments of the disclosure include cells that express a CAR (i.e, CARTS).
• the cell can be of any kind, including an immune cell that can express the CAR for cancer therapy or a cell, such as a bacterial cell, that harbors an expression vector that encodes the CAR.
• a CAR i.e, CARTS
• the terms “cell,” “cell line,” and “cell culture” can be used
• host cell can refer to a eukaryotic cell that is can replicate a vector and/or expressing a heterologous gene encoded by a vector.
• a host cell can, and has been, used as a recipient for vectors.
• a host cell can be "transfected” or “transformed,” which can refer to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
• a transformed cell includes the primary subject cell and its progeny.
• a host cell is a T cell, including a cytotoxic T cell (also known as TC, Cytotoxic T Lymphocyte, CTL, T-Killer cell, cytolytic T cell, CD8+ T-cells or killer T cell); NK cells and NKT cells are also encompassed in the disclosure.
• cytotoxic T cell also known as TC, Cytotoxic T Lymphocyte, CTL, T-Killer cell, cytolytic T cell, CD8+ T-cells or killer T cell
• NK cells and NKT cells are also encompassed in the disclosure.
• Some vectors can employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells.
• One of skill in the art would further understand the conditions under which to incubate all of the above described host cells to maintain them and to permit replication of a vector.
• Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.
• the cells can be autologous cells, syngeneic cells, allogenic cells and even in some cases, xenogeneic cells.
• the invention further includes CARTs that are modified to secrete one or more polypeptides.
• CARTs can be referred to as CART factories, CAR T cell factories, or armed CARTs.
• the polypeptide can be, for example, an antibody or fragment thereof as described herein.
• the polypeptide can be an antibody or cytokine.
• the antibody is specific for TIGIT, CAIX, GITR, PD-L1, PD-L2.
• PD-1, CCR4, CTLA-4, VISTA, or CD70 can secrete PD-L1 mAbs locally at the tumor site to restore the effective anti-cancer immunity and/or reverse T cell exhaustion.
• the armed CART secretes IL-12, IL-15, or IL-18.
• a second expression construct which can be in the same DNA vector as that which encodes the CAR (e.g. the antigen-recognition domain) or in a second separate vector, can be used to encode a mini body (scFv-Fc) or antibody, or a fragment thereof, that is directed against a single or multiple antigens of interest, and can be cloned after an internal ribosomal entry site (IRES).
• the second expression cassette comprises either a fluorescent molecule or an immune-modulating minibody.
• TIGIT is a T-cell coinhibitory receptor that limits antitumor and other T-cell dependent chronic immune responses, such as CD8+ T cell-dependent immune responses.
• TIGIT is expressed on subsets of activated T cells and natural killer (NK) cells.
• NK natural killer
• TIGIT is highly expressed on tumor-infiltrating T-cells. In cancer models, antibody blockade of TIGIT contributed to enhanced CD8+ T cell effector function and tumor clearance.
• the anti-TIGIT antibody of the armed CART comprises one or more of the anti-TIGIT antibody clones (or fragments thereof, such as FR1, FR2, FR3, FR4, CDR1, CDR2, CDR3, or any combinations of the framework and/or CDR regions therein) described in FIG.81.
• the anti-TIGIT antibody can comprise a CDR1 of the VH region of having the sequence: GYTF... TSYG (SEQ ID NO:[ ]); CDR2 of the VH region having the sequence: ISAY..NGNT (SEQ ID NO:[ ]); CDR3 of the VH region having the sequence: ARDPGLWF GLTHDYYFD Y (SEQ ID NO:[ ]); CDR1 of the VL region having the sequence SSNF...GSNT (SEQ ID NO:[ ]); CDR2 of the VL region having the sequence:
• the anti-TIGIT antibody can comprise a CDR1 of the VH region of having the sequence: GFTF....SDYS (SEQ ID NO:[ ]); CDR2 of the VH region having the sequence: INSD..GSRT (SEQ ID NO:[ ]); CDR3 of the VH region having the sequence: ARGPGFFGFDI (SEQ ID NO:[ ]); CDR1 of the VL region having the sequence
• the anti-TIGIT antibody can comprise a CDR1 of the VH region of having the sequence: GYSF....TNYW (SEQ ID NO:[ ]); CDR2 of the VH region having the sequence: INPV..NSRT (SEQ ID NO:[ ]); CDR3 of the VH region having the sequence: ARYYYYAMEV (SEQ ID NO:[ ]); CDR1 of the VL region having the sequence
• the anti-TIGIT antibody can comprise a CDR1 of the VH region of having the sequence: GYTF....TNYG (SEQ ID NO:[ ]); CDR2 of the VH region having the sequence: VDNN..NGNI (SEQ ID N0:[ ]); CDR3 of the VH region having the sequence: ARGLFSSRWYLWFDP (SEQ ID NO:[ ]); CDR1 of the VL region having the sequence
• the anti-TIGIT antibody can comprise a CDR1 of the VH region of having the sequence: GGTF....SSYA (SEQ ID NO:[ ]); CDR2 of the VH region having the sequence: ILPM..FGST (SEQ ID NO:[ ]); CDR3 of the VH region having the sequence: ARGRDIVAPSNSGFDV (SEQ ID NO:[ ]); CDR1 of the VL region having the sequence
• armed CARTS can secrete anti-PDLl antibodies or fragments thereof.
• the armed CARTS can secrete anti-PDLl antibodies or fragments thereof disclosed in provisional patent application no. 62/624,455, which is incorporated by reference herein in its entirety.
• Exemplary anti-PDLl antibodies include antibodies having a VH amino acid sequence having SEQ ID NO: [ ] and/or a VL amino acid sequence having SEQ ID NO: [ ]. See FIG. 78 and FIG. 81, for example.
• the anti-PDLl antibodies of the armed CART comprises on or more of the anti-PDLl antibody clones (or fragments thereof, such as FR1, FR2, FR3,
• the anti-PDLl antibodies have a heavy chain with three CDRs including the amino acid sequences SEQ ID NO: [ ], [ ], and/or [ ], respectively and a light chain with three CDRs including the amino acid sequence.
• armed CARTS can secrete anti -PD 1 antibodies or fragments thereof.
• Exemplary anti -PD 1 antibodies include antibodies having a VH amino acid sequence having SEQ ID NO: [ ] and a VL amino acid sequence having SEQ ID NO: [ ]. See FIG. 78 and FIG. 83, for example.
• the anti-PDl antibodies of the armed CART comprises on or more of the anti-PDl antibody clones (or fragments thereof, such as FR1, FR2, FR3, FR4,
• the anti-PDl antibodies have a heavy chain with three CDRs including the amino acid sequences SEQ ID NO: [ ], [ ], and/or [ ], respectively and a light chain with three CDRs including the amino acid sequences SEQ ID NO: [ ], [ ], and/or [ ], respectively and a light chain with three CDRs including the amino acid sequences SEQ ID NO: [ ], [ ], and/or [ ], respectively and a light chain with three CDRs including the amino acid sequences SEQ ID NO: [ ], [ ], and/or [ ], respectively and a light chain with three CDRs including the amino acid sequences SEQ ID NO: [ ], [ ], and/or [ ], respectively and a light chain with three CDRs including the amino acid sequences SEQ ID NO: [ ], [ ], and/or [ ], respectively and a light chain with three CDRs including the amino acid sequences SEQ ID NO: [ ], [ ], and/or [ ], respectively and a light chain with three CDRs
• amino acid sequences of the heavy and light chain complementary determining regions of the PD-1 antibodies are shown below:
• VH Heavy chain (VH) complementary determining regions (CDRs) of the PD-1 antiboc ies
• VL Light chain
• CDRs complementary determining regions
• armed CARTS can secrete anti-CCR4 antibodies or fragments thereof.
• armed CARTS can secrete anti-CCR4 antibodies or fragments as described in W02009/086514, WO 2013/166500, PCT/US2015/054202, or PCT/US2016/026232.
• CART can comprise an antibody having a VH CDR1 region having the amino acid sequence GYTFASYY (SEQ ID NO: [ ]); a VH CDR2 region having the amino acid sequence WINP GNVNTKYNEKFKG (SEQ ID NO: [ ]); a VH CDR3 region having the amino acid sequence STYYRPLDY (SEQ ID NO: [ ]); and/or VL CDR1 region having amino acid sequence KSSQSILYSSNQKNYLA (SEQ ID NO: [ ]); a VL CDR2 region having the amino acid sequence WASTRES (SEQ ID NO: [ ]) and/or a VL CDR3 region having the amino acid sequence HQYLSSYT (SEQ ID NO: [ ]).
• the anti-CCR4 protein or fragment thereof of the armed CAR can comprise an antibody having a VH amino acid sequence
• CART can comprise an antibody having a:
• VH chain of antibody 1-44 (SEQ ID NO: [ ])
• VL chain of antibody 1-44 (SEQ ID NO: [ ])
• VH chain of antibody 1-49 (SEQ ID NO: [ ])
• VH chain of antibody 2-1 (SEQ ID NO: [ ])
• VL chain of antibody 2-1 (SEQ ID NO: [ ])
• VH chain of antibody 2-2 (SEQ ID NO: [ ])
• VL chain of antibody 2-2 (SEQ ID NO: [ ])
• VH chain of antibody 2-3 (SEQ ID NO: [ ])
• VL chain of antibody 2-3 (SEQ ID NO: [ ])
• Armed CART can be constructed by including a nucleic acid encoding the polypeptide of interest after the intracellular signaling domain.
• an internal ribosome entry site (IRES)
• IRES internal ribosome entry site
• the methods and compositions presented herein provide a target-specific T cell, such as a T cell with specificity for CAIX and/or CD70, armed with the power to secrete polypeptides in the tumor microenvironment, for example to combat T cell exhaustion.
• a target-specific T cell such as a T cell with specificity for CAIX and/or CD70
• MDSCs myeloid-derived suppressor cells
• MDSCs are a heterogeneous population of early myeloid progenitors, immature granulocytes, macrophages, and dendritic cells at different stages of differentiation that comprise the tumor microenvironment.
• MDSCs are induced by pro-inflammatory cytokines and are found in increased numbers in infectious and inflammatory disease conditions. Without wishing to be bound by theory, their presence in the tumor microenvironment indicates a causative role in promoting tumor- associated immune suppression.
• Human MDSCs express Siglec-3/CD33 (GENBANK Accession No. NM_001772.4 (for nucleotide sequence) and NP_001763.3 (for amino acid sequence) and have heterogeneous expression of CD14 (GENBANK Accession No.
• NM_000591.4 for nucleotide sequence
• NP_000582.1 for amino acid sequence
• MDSC markers useful for identifying MDSCs include, but are not limited to B7-1/CD8 (NM_001145873.1 (for nucleotide sequence), NP_001139345.1 (for amino acid sequence)), CCR2(NM_001123041.2 (for nucleotide sequence), NP_001116513.2 (for amino acid sequence)), CDld (NM_001766.3 (for nucleotide sequence), NP_001757.1 (for amino acid sequence)), CD2 (NM_001328609.2 (for nucleotide sequence), NP_001315538.1 (for amino acid sequence)), CD31/PECAM-1 (NM_000442.5 (for nucleotide sequence), NP_000433.4 (for amino acid sequence)), CD43 (NM_001030288.3 (for nucleotide
• the methods and compositions presented herein provide a target-specific T cell, such as a T cell with specificity for CAIX and/or CD70, armed with the power to secrete polypeptides that target MDSCs in the tumor microenvironment.
• a target-specific T cell such as a T cell with specificity for CAIX and/or CD70
• the secreted polypeptides can target one or more MDSC markers (e.g., CD33, CD14, and/or CD15, or other MDSC markers listed herein).
• Expression vectors that encode the CARs can be introduced as one or more DNA molecules or constructs, where there may be at least one marker that will allow for selection of host cells that contain the construct(s).
• constructs can be prepared in conventional ways, where the genes and regulatory regions can be isolated, as appropriate, ligated, cloned in an appropriate cloning host, analyzed by restriction or sequencing, or other convenient means. Particularly, using PCR, individual fragments including all or portions of a functional unit can be isolated, where one or more mutations may be introduced using "primer repair", ligation, in vitro
• construct(s) once completed and demonstrated to have the appropriate sequences may then be introduced into the CTL by any convenient means.
• the constructs may be integrated and packaged into non-replicating, defective viral genomes like Adenovirus, Adeno-associated virus (AAV), or Herpes simplex virus (HSV) or others, including retroviral vectors or lentiviral vectors, for infection or transduction into cells.
• the constructs may include viral sequences for transfection.
• the construct may be introduced by fusion, electroporation, biolistics, transfection, lipofection, or the like.
• the host cells can be grown and expanded in culture before introduction of the construct(s), followed by the appropriate treatment for introduction of the construct(s) and integration of the construct(s).
• the cells are then expanded and screened by virtue of a marker present in the construct.
• markers that may be used successfully include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, etc.
• a target site for homologous recombination where a construct be integrated at a particular locus.
• .OMEGA O-vectors. See, for example, Thomas and Capecchi, Cell (1987) 51, 503-512; Mansour, et al, Nature (1988) 336, 348-352; and Joyner, et al., Nature (1989) 338, 153-156.
• the constructs can be introduced as a single DNA molecule encoding at least the CAR and optionally another gene, or different DNA molecules having one or more genes.
• Other genes include genes that encode therapeutic molecules or suicide genes, for example.
• the constructs may be introduced simultaneously or consecutively, each with the same or different markers.
• Vectors containing useful elements such as bacterial or yeast origins of replication, selectable and/or amplifiable markers, promoter/enhancer elements for expression in prokaryotes or eukaryotes, etc. that may be used to prepare stocks of construct DNAs and for carrying out transfections are well known in the art, and many are commercially available.
• aspects of the disclosure are directed towards methods of treating a subject afflicted with a cancer.
• aspects of the disclosure are directed towards methods of killing a cancer cell, such as a renal cancer cell.
• a cancer cell such as a renal cancer cell.
• B7-GGGGS-G36 CAR T cells had more killing activity on the targeted renal carcinoma cells (CAIX+ CD70+) than non-targeted cells (CAIX+ CD70-, CAIX- CD70+, CAIX- CD70-). Further, referring to FIG. 12, for example, B7-GGGGS-G36 CAR T cells had more killing activity on the targeted renal carcinoma cells (CAIX+ CD70+) than non-targeted cells (CAIX+ CD70-, CAIX- CD70+, CAIX- CD70-). Further, referring to
• FIG. 57 for example, bispecific split CAR achieved superior killing when compared to mono CAR or bispecific CARs.
• aspects of the disclosure are further directed towards methods of stopping or reducing progression or promoting regression of a cancer in a subject.
• aspects of the disclosure are directed towards a method of reducing cellular proliferation of a cancer cell in a subject. See, for example, FIG. 80.
• Cancer and “cancerous” can refer to or describe, for example, the physiological condition in mammals that is characterized by unregulated cell growth.
• cancer examples include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, smallcell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer.
• the cancer is renal cell carcinoma, such as ccRCC.
• TME tumor microenvironment
• ECM extracellular matrix
• the secretion of a mono, bi-, or tri-specific minibody, antibody or minibody/antibody fusion protein by the CAR-T cell at the tumor site could provide additional benefit by altering (i.e., modulating) the immune-repressive tumor microenvironment.
• the method comprises administering to a subject afflicted with a cancer a therapeutically effective amount of an engineered cell as described herein.
• Therapeutically effective amounts can depend on the severity and course of the cancer, previous therapy, the subject's health status, weight, and response to the drugs, and the judgment of the treating physician.
• the subject can be afflicted with cancer such as liquid cancers (i.e., blood cancers) and/or solid cancers (i.e., tumors).
• cancer can be benign or malignant, and can be one that is influenced by the immune system.
• Embodiments as described herein can modulate the immune system so as to treat the subject afflicted with cancer.
• “Modulating” can refer to up-regulation, induction, stimulation, potentiation, and/or relief of inhibition, as well as inhibition, attenuation and/or down-regulation or suppression.
• the activity of the subject's immune system is modulated, the microenvironment surround the cancer cell and/or tumor is modulated, or both.
• embodiments as described herein can alter the immune-repressive tumor microenvironment, reducing the microenvironment-dependent immune suppression, so as to modulate (or allow) the immune system to kill tumor cells.
• One embodiment is directed towards methods of treating a subject afflicted with renal cell carcinoma.
• Immune therapies such as those described herein, offer an exciting therapeutic option for RCC.
• embodiments comprise engineering a chimeric- antigen receptor (CAR) T-cell for RCC.
• An "individual" or “subject” can be a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.
• the cells according to the disclosure can be used for treating cancer in a patient in need thereof.
• said isolated cell according to the invention can be used in the manufacture of a medicament for treatment of a cancer, viral infections of autoimmune disorders, in a patient in need thereof.
• the present disclosure can rely on methods for treating patients in need thereof, said method comprising at least one of the following steps: (a) providing a chimeric antigen receptor cells according to the invention and (b) administrating the cells to said patient.
• Said treatment can be ameliorating, curative or prophylactic. It can be either part of an autologous immunotherapy or part of an allogenic immunotherapy treatment.
• autologous it is meant that cells, cell line or population of cells used for treating patients are originating from said patient or from a Human Leucocyte Antigen (HLA) compatible donor.
• HLA Human Leucocyte Antigen
• allogeneic is meant that the cells or population of cells used for treating patients are not originating from said patient but from a donor.
• the invention is particularly suited for allogenic immunotherapy, insofar as it allows the transformation of T-cells, obtained from donors, into non-alloreactive cells. This may be done under standard protocols and reproduced as many times as needed.
• the resulted modified T cells may be pooled and administrated to one or several patients, being made available as an "off the shelf therapeutic product.
• Cancers that can be used with the disclosed methods are described in the previous section. Said treatment can be used to treat patients diagnosed with cancer. Cancers that may be treated include tumors that are not vascularized, or not yet substantially vascularized, as well as vascularized tumors. The cancers may comprise nonsolid tumors (such as
• hematological tumors for example, leukemias and lymphomas
• hematological tumors for example, leukemias and lymphomas
• solid tumors for example, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid
• malignancies benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas.
• malignancies e.g., sarcomas, carcinomas, and melanomas.
• adult tumors/cancers and pediatric tumors/cancers are also included.
• It can be a treatment in combination with one or more therapies against cancer selected from the group of antibodies therapy, chemotherapy, cytokines therapy, dendritic cell therapy, gene therapy, hormone therapy, laser light therapy and radiation therapy.
• said treatment can be administrated into patients undergoing an immunosuppressive treatment.
• the present invention uses cells or population of cells, which have been made resistant to at least one immunosuppressive agent due to the inactivation of a gene encoding a receptor for such immunosuppressive agent.
• the immunosuppressive treatment should help the selection and expansion of the T-cells according to the invention within the patient.
• the cell compositions of the present invention are administered to a patient in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAM PATH.
• chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAM PATH.
• the cell compositions of the present invention are administered following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan.
• subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell
• modified cells obtained by any one of the methods described here can be used in a particular aspect of the invention for treating patients in need thereof against Host versus Graft (HvG) rejection and Graft versus Host Disease (GvHD); therefore in the scope of the present invention is a method of treating patients in need thereof against Host versus Graft (HvG) rejection and Graft versus Host Disease (GvHD) comprising treating said patient by administering to said patient an effective amount of modified cells comprising inactivated TCR alpha and/or TCR beta genes.
• the disclosure is particularly suited for allogenic immunotherapy, insofar as it allows the transformation of T-cells, obtained from donors, into non-alloreactive cells. This can be done under standard protocols and reproduced as many times as needed.
• the resulted modified T cells may be pooled and administrated to one or several patients, being made available as an "off the shelf therapeutic product.
• the cells can be introduced into a host organism, e.g. a mammal, in a wide variety of ways.
• the cells can be introduced at the site of the tumor, in specific embodiments, although in alternative embodiments the cells hone to the cancer or are modified to hone to the cancer.
• the number of cells that are employed will depend upon a number of circumstances, the purpose for the introduction, the lifetime of the cells, the protocol to be used, for example, the number of administrations, the ability of the cells to multiply, the stability of the recombinant construct, and the like.
• the cells may be applied as a dispersion, for example, being injected at or near the site of interest.
• the cells may be in a physiologically-acceptable medium.
• the cells are encapsulated to inhibit immune recognition and placed at the site of the tumor.
• the cells can be administered as desired. Depending upon the response desired, the manner of administration, the life of the cells, the number of cells present, various protocols may be employed. The number of administrations will depend upon the factors described above at least in part.
• the administration of the cells or population of cells according to the present invention can be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation.
• the compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally.
• the cell compositions of the present invention are administered by intravenous injection.
• the administration of the cells or population of cells can consist of the administration of 104 -109 cells per kg body weight, for example, 105 to 106 cells/kg body weight including all integer values of cell numbers within those ranges.
• the cells or population of cells can be administrated in one or more doses.
• said effective amount of cells are administrated as a single dose.
• said effective amount of cells are administrated as more than one dose over a period time. Timing of administration is within the judgment of managing physician and depends on the clinical condition of the patient.
• the cells or population of cells may be obtained from any source, such as a blood bank or a donor.
• An effective amount means an amount which provides a therapeutic or prophylactic benefit.
• the dosage administrated will be dependent upon the age, health and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired.
• the system is subject to many variables, such as the cellular response to the ligand, the efficiency of expression and, as appropriate, the level of secretion, the activity of the expression product, the particular need of the patient, which may vary with time and circumstances, the rate of loss of the cellular activity as a result of loss of cells or expression activity of individual cells, and the like. Therefore, for each individual patient, even if there were universal cells which could be administered to the population at large, each patient would be monitored for the proper dosage for the individual, and such practices of monitoring a patient are routine in the art.
• the CARs of the disclosure can be expressed from an expression vector.
• DNA constructs which can also be referred to as "DNA vectors", as described herein, can be cloned into a vector which will be used to transduce and produce chimeric- antigen receptor T-cells that secrete polypeptides and/or fragments thereof.
• DNA constructs can be cloned into a lentiviral vector for production of lentivirus, which will be used to transduce and produce chimeric-antigen receptor T-cells that secrete a mono, bi- or tri-specific immune-modulating antibody/minibody and/or antibody-fusion protein at the tumor site.
• vector can refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated.
• a nucleic acid sequence can be "exogenous,” which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found.
• Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs).
• YACs artificial chromosomes
• expression vector refers to any type of genetic construct comprising a nucleic acid coding for a RNA that can be transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes.
• Expression vectors can contain a variety of "control sequences,” which refer to nucleic acid sequences necessary for the transcription and translation of an operably linked coding sequence in a particular host cell. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra.
• a "promoter” is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It can contain genetic elements at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors, to initiate the specific transcription a nucleic acid sequence.
• the phrases "operatively positioned,” “operatively linked,” “under control,” and “under transcriptional control” mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and/or expression of that sequence.
• a promoter comprises a sequence that functions to position the start site for RNA synthesis.
• the best known example of this is the TATA box, but in some promoters lacking a TATA box, such as, for example, the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation. Additional promoter elements regulate the frequency of transcriptional initiation. These are located in the region 30 110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well.
• a coding sequence "under the control of' a promoter, one positions the 5' end of the transcription initiation site of the transcriptional reading frame "downstream" of (i.e., 3' of) the chosen promoter.
• the "upstream” promoter stimulates transcription of the DNA and promotes expression of the encoded RNA.
• the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.
• the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.
• individual elements can function either cooperatively or independently to activate transcription.
• a promoter may or may not be used in conjunction with an "enhancer,” which refers to a cis- acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.
• a promoter can be one naturally associated with a nucleic acid sequence, as may be obtained by isolating the 5 prime' non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as "endogenous.”
• an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence.
• certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment.
• a recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment.
• Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other virus, or prokaryotic or eukaryotic cell, and promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression.
• promoters that are most commonly used in recombinant DNA construction include the lactamase (penicillinase), lactose and tryptophan (trp) promoter systems.
• sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR.TM., in connection with the compositions disclosed herein (see U.S. Pat. Nos. 4,683,202 and 5,928,906, each incorporated herein by reference).
• control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.
• promoter and/or enhancer that effectively directs the expression of the DNA segment in the organelle, cell type, tissue, organ, or organism chosen for expression.
• Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression, (see, for example Sambrook et al. 1989, incorporated herein by reference).
• the promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides.
• the promoter may be heterologous or endogenous.
• any promoter/enhancer combination could also be used to drive expression.
• Use of a T3, T7 or SP6 cytoplasmic expression system is another embodiment.
• Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.
• tissue-specific promoters or elements as well as assays to characterize their activity, is well known to those of skill in the art.
• a specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily determine this and providing the necessary signals
• IVS internal ribosome entry sites
• Vectors can include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector.
• MCS multiple cloning site
• Restriction enzyme digestion refers to catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at specific locations in a nucleic acid molecule. Many of these restriction enzymes are commercially available. Use of such enzymes is widely understood by those of skill in the art. Frequently, a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to allow exogenous sequences to be ligated to the vector.
• Ligand refers to the process of forming phosphodiester bonds between two nucleic acid fragments, which may or may not be contiguous with each other. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology.
• Splicing sites may also be employed.
• a plasmid vector can be used to transform a host cell. Plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences which can provide phenotypic selection in transformed cells.
• E. coli is often transformed using derivatives of pBR322, a plasmid derived from an E. coli species.
• pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells.
• the pBR plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, for example, promoters which can be used by the microbial organism for expression of its own proteins.
• phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts.
• the phage lambda GEM.TM. 11 may be utilized in making a recombinant phage vector which can be used to transform host cells, such as, for example, E. coli LE392.
• Further useful plasmid vectors include pIN vectors (Inouye et al, 1985); and pGEX vectors, for use in generating glutathione S transferase (GST) soluble fusion proteins for later purification and separation or cleavage.
• GST glutathione S transferase
• Other suitable fusion proteins are those with galactosidase, ubiquitin, and the like.
• Bacterial host cells for example, E. coli, comprising the expression vector, are grown in any of a number of suitable media, for example, LB.
• suitable media for example, LB.
• the expression of the recombinant protein in certain vectors may be induced, as would be understood by those of skill in the art, by contacting a host cell with an agent specific for certain promoters, e.g., by adding IPTG to the media or by switching incubation to a higher temperature. After culturing the bacteria for a further period, for example, between 2 and 24 h, the cells are collected by centrifugation and washed to remove residual media.
• Components of the present invention may be a viral vector that encodes one or more CARs of the invention.
• Non-limiting examples of virus vectors that may be used to deliver a nucleic acid of the present invention are described below.
• a particular method for delivery of the nucleic acid involves the use of an adenovirus expression vector.
• adenovirus vectors are known to have a low capacity for integration into genomic DNA, this feature is counterbalanced by the high efficiency of gene transfer afforded by these vectors.
• "Adenovirus expression vector” is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to ultimately express a tissue or cell specific construct that has been cloned therein.
• Knowledge of the genetic organization or adenovirus, a 36 kb, linear, double stranded DNA virus allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus and Horwitz, 1992).
• the nucleic acid may be introduced into the cell using adenovirus assisted transfection. Increased transfection efficiencies have been reported in cell systems using adenovirus coupled systems (Kelleher and Vos, 1994; Cotten et al, 1992; Curiel, 1994).
• Adeno associated virus (AAV) is an attractive vector system for use in the cells of the present invention as it has a high frequency of integration and it can infect nondividing cells, thus making it useful for delivery of genes into mammalian cells, for example, in tissue culture (Muzyczka, 1992) or in vivo.
• AAV has a broad host range for infectivity (Tratschin et al, 1984; Laughlin et al., 1986; Lebkowski et al, 1988; McLaughlin et al, 1988). Details concerning the generation and use of rAAV vectors are described in U.S. Pat. Nos. 5,139,941 and 4,797,368, each incorporated herein by reference.
• Retroviruses are useful as delivery vectors because of their ability to integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and of being packaged in special cell lines (Miller, 1992).
• a nucleic acid e.g., one encoding the sequence of interest
• a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed (Mann et al, 1983).
• Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al, 1975).
• Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. Lentiviral vectors are well known in the art (see, for example, Naldini et al., 1996; Zufferey et al, 1997; Blomer et al, 1997; U.S. Pat. Nos. 6,013,516 and 5,994,136). Some examples of lentivirus include the Human Immunodeficiency Viruses: HIV-1, HIV -2 and the Simian Immunodeficiency Virus: SIV. Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe.
• Recombinant lentiviral vectors can infect non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression of nucleic acid sequences.
• recombinant lentivirus can infect a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in U.S. Pat. No. 5,994,136, incorporated herein by reference.
• One may target the recombinant virus by linkage of the envelope protein with an antibody or a particular ligand for targeting to a receptor of a particular cell-type.
• a sequence (including a regulatory region) of interest into the viral vector, along with another gene which encodes the ligand for a receptor on a specific target cell, for example, the vector is now target-specific.
• viral vectors may be employed as vaccine constructs in the present invention.
• Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988), Sindbis virus, cytomegalovirus and herpes simplex virus may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988;
• a nucleic acid to be delivered may be housed within an infective virus that has been engineered to express a specific binding ligand.
• the virus particle will thus bind specifically to the cognate receptors of the target cell and deliver the contents to the cell.
• a new approach designed to allow specific targeting of retrovirus vectors was developed based on the chemical modification of a retrovirus by the chemical addition of lactose residues to the viral envelope. This modification can permit the specific infection of hepatocytes via sialogly coprotein receptors.
• Suitable methods for nucleic acid delivery for transfection or transformation of cells are known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by ex vivo transfection, by injection, and so forth.
• cells may be stably or transiently transformed.
• cells or tissues may be removed and transfected ex vivo using nucleic acids of the present invention.
• the transplanted cells or tissues may be placed into an organism.
• a nucleic acid is expressed in the transplanted cells.
• compositions described herein may be comprised in a kit.
• one or more cells for use in cell therapy and/or the reagents to generate one or more cells for use in cell therapy that harbors recombinant expression vectors may be comprised in a kit.
• the kit components are provided in suitable container means.
• kits may be packaged either in aqueous media or in lyophilized form.
• the container means of the kits can include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and suitably aliquoted. Where there are more than one component in the kit, the kit also can contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial.
• the kits of the present invention also can include a means for containing the components in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.
• the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly useful.
• the container means may itself be a syringe, pipette, and/or other such like apparatus, from which the formulation may be applied to an infected area of the body, injected into an animal, and/or even applied to and/or mixed with the other components of the kit.
• the components of the kit may be provided as dried powder(s).
• kits When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent.
• the solvent may also be provided in another container means.
• the kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and/or other diluent.
• kits that are to be used for cell therapy are provided in a kit, and in some cases the cells are essentially the sole component of the kit.
• the kit may comprise reagents and materials to make the desired cell.
• the reagents and materials include primers for amplifying desired sequences, nucleotides, suitable buffers or buffer reagents, salt, and so forth, and in some cases the reagents include vectors and/or DNA that encodes a CAR as described herein and/or regulatory elements therefor.
• kits suitable for extracting one or more samples from an individual.
• the apparatus may be a syringe, scalpel, and so forth.
• the kit in addition to cell therapy
• kits also includes a second cancer therapy, such as chemotherapy, hormone therapy, and/or immunotherapy, for example.
• the kit(s) may be tailored to a particular cancer for an individual and comprise respective second cancer therapies for the individual.
• an "anti-cancer” agent can negatively affect cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer.
• these other compositions would be provided in a combined amount effective to kill or inhibit proliferation of the cell. This process may involve contacting the cancer cells with the expression construct and the agent(s) or multiple factor(s) at the same time. This may be achieved by contacting the cell with a single composition or
• pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the expression construct and the other includes the second agent(s).
• Tumor cell resistance to chemotherapy and radiotherapy agents represents a major problem in clinical oncology.
• One goal of current cancer research is to find ways to improve the efficacy of chemo- and radiotherapy by combining it with other therapies.
• cell therapy could be used similarly in conjunction with chemotherapeutic, radiotherapeutic, or immunotherapeutic intervention, as well as pro-apoptotic or cell cycle regulating agents.
• the present inventive therapy may precede or follow the other agent treatment by intervals ranging from minutes to weeks.
• the other agent and present invention are applied separately to the individual, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and inventive therapy would still be able to exert an advantageously combined effect on the cell.
• one may contact the cell with both modalities within about 12-24 h of each other (for example, within about 6-12 h of each other).
• treatment cycles would be repeated as necessary.
• various standard therapies, as well as surgical intervention may be applied in combination with the inventive cell therapy.
• Cancer therapies also include a variety of combination therapies with both chemical and radiation based treatments.
• Combination chemotherapies include, but are not limited to, for example, abraxane, altretamine, docetaxel, herceptin, methotrexate, novantrone, zoladex, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, famesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristin,
• chemotherapy for the individual is employed in conjunction with the invention, for example before, during and/or after administration of the invention
• DNA damaging factors include what are commonly known as .gamma. -rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells.
• Other forms of DNA damaging factors are also useful such as microwaves and UV-irradiation. It is most likely that all of these factors effect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes.
• Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.
• Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
• Immunotherapeutics rely on the use of immune effector cells and molecules to target and destroy cancer cells.
• the immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell.
• the antibody alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing.
• the antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent.
• the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target.
• Various effector cells include cytotoxic T cells and NK cells.
• Immunotherapy other than the inventive therapy described herein could thus be used as part of a combined therapy, in conjunction with the present cell therapy.
• the approach for combined therapy is discussed herein.
• the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present invention.
• Common tumor markers include PD-1, PD-L1, CTLA4, carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and pi 55.
• the secondary treatment is a gene therapy in which a therapeutic polynucleotide is administered before, after, or at the same time as the present invention clinical embodiments.
• a variety of expression products are encompassed within the invention, including inducers of cellular proliferation, inhibitors of cellular proliferation, or regulators of programmed cell death.
• Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapies.
• Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed.
• Tumor resection refers to physical removal of at least part of a tumor.
• treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs' surgery).
• the present invention may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.
• a cavity may be formed in the body.
• Treatment may be accomplished by perfusion, direct injection or local application of the area with an additional anti-cancer therapy.
• Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.
• These treatments may be of varying dosages as well.
• other agents may be used in combination with the present invention to improve the therapeutic efficacy of treatment.
• additional agents include immunomodulatory agents, agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, or agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers.
• Immunomodulatory agents include tumor necrosis factor; interferon alpha, beta, and gamma; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-1, MIP-lbeta, MCP-1, RANTES, and other chemokines.
• upregulation of cell surface receptors or their ligands such as Fas/Fas ligand, DR4 or DR5 /TRAIL would potentiate the apoptotic inducing abilities of the present invention by establishment of an autocrine or paracrine effect on hyperproliferative cells. Increases intercellular signaling by elevating the number of GAP junctions would increase the anti -hyperproliferative effects on the neighboring hyperproliferative cell population.
• cytostatic or differentiation agents can be used in combination with the present invention to improve the anti-hyperproliferative efficacy of the treatments.
• Inhibitors of cell adhesion can also be used to improve the efficacy of the present invention.
• cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin.
• FAKs focal adhesion kinase
• Lovastatin Lovastatin.
• other agents that increase the sensitivity of a hyperproliferative cell to apoptosis such as the antibody c225, could be used in combination with the present invention to improve the treatment efficacy.
• aspects of the disclosure are further directed towards methods of and kits for assessing the killing capabilities of engineered CAR T cells.
• embodiments are directed towards an immune complex analysis method and kits to determine the CAR T cell activity during co-culture with cancer cells.
• target cancer cells for example, HEK293T, MDA-MB-231, MDA-MB-468, HCC38, and skrc59
• a dye for example, ViaStainTM Tracer Blue dye
• T cell types for example, two different T cell types
• E:T effector-to-target
• co-culture for example, 24 hours
• the plate is scanned and analyzed (for example, using the bright field and blue fluorescent channels).
• the immune complexes were analyzed by confluence measurement and compared to the negative control of untransduced T cells. As a result, data plots displayed the CAR T cell activities for all of tested target and effector cells
• Utilization of an image cytometry platform can visually confirm interactions between effector and target cells, thus making results highly accurate and robust.
• Clear cell renal cell carcinoma is the major type of RCC which is among the 10 most common cancers in both men and women.
• Chimeric Antigen Receptor (CAR) T cells have proven to be a powerful, clinically translatable immunotherapy for hematologic malignancies. However, these results have not been translatable to solid tumors due to inefficient homing of CAR T cells, the suppressive tumor microenvironment, and on- target off-tumor toxicities resulted from the sharing of CAR T targeting epitopes on healthy tissues.
• immune checkpoint blockade has shown an enhanced effect on antitumor response by restoring the local antitumor immunity.
• CAR T cell factories were designed to empower CAR T cells through the secretion of human anti-immune checkpoint inhibitor monoclonal antibodies (mAbs) locally at the tumor site.
• mAbs human anti-immune checkpoint inhibitor monoclonal antibodies
• CAIX is an ideal target for ccRCC therapy and used as a CAR target for the first clinical trial (see, for example, Lamers, Sleijfer et al. 2006; Lamers, Willemsen et al. 2011).
• second generation CARs were developed by introducing a 2nd targeting scFv (for example, anti-CD70 scFv) in the CAR T cell factory together with the first targeting scFv (for example, anti-CAIX scFv) to allow the CAR to target two unique antigens simultaneously. See, for example, FIG. 1.
• a 2nd targeting scFv for example, anti-CD70 scFv
• first targeting scFv for example, anti-CAIX scFv
• CD28, 41BB costimulatory domain
• CD3 activating domain
• the 4th generation lentivirus packaging system was used to obtain CAR lentiviruses and primary T cells isolated from PBMCs were transduced to express dual CAR and were tested against different cell lines in vitro.
• a humanized orthotopic ccRCC mouse model was established by injecting luciferized ccRCC cells under the kidney capsule ofNSG-SGM3 mice with a reconstituted human immune system.
• the 2nd generation CAR T was discovered.
• the 2nd generation CAR T cell factories can be used for the treatment of cancers, such as ccRCC, and are able to eliminate side effects on normal tissues.
• Chimeric Antigen Receptor (CAR) T cells have proven to be a powerful immunotherapy for hematologic malignancies, however, these have not been translated to solid tumors.
• Our CAR T cell factories empower CAR T cells through secreting antibodies, such as human anti-immune checkpoint inhibitor monoclonal antibodies (mAbs), locally at the tumor site to restore tumor microenvironment to achieve solid tumor cure.
• mAbs human anti-immune checkpoint inhibitor monoclonal antibodies
• the 2nd generation CAR has elevated efficacy and safety and holds great promise in clinic.
• the bispecific tandem CAR was designed to target antigens associated with tumors, such as CAIX and CD70 (see FIG. 1 and FIG. 2, for example) which are both highly expressed and co-expressed on ccRCC primary cell by IHC (FIG. 3).
• Successful panning of a 27 billion member phage display library has led to the identification of several anti-CD70 scFvs (see FIG. 6, for example).
• the anti-CAIX scFvs we discovered before and anti-CD70 scFvs were cloned into pHAGE vector with different linkers (See FIG. 9, for example). Then lentiviruses were packaged and transduced with primary T cells.
• CAR T cells were obtained and evaluated on 4 different CRISPR engineered cell lines (see FIG. 5, for example). The killing activity was assessed in Celigo and Cr51 release assay (See FIG. 12 and 13, for example). Selectivity was shown as described herein.
• This CAR T cell factories can be used in therapy of CAIX and/or CD70 over expressed cancers or in combination with other therapies.

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