Patent Application
20210092942
The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: Hera sequence listing_ST25.TXT, date recorded: Mar. 26, 2018, file size 4 kilobytes).
Animal models of human cancer offer the potential to study human tumor growth kinetics, genetic variance among human cancers, and provide in vivo platforms for drug efficacy testing. Immunodeficient mouse models have been invaluable in modeling a wide range of human cancers and testing drug efficacy. However, the use of mouse models is limited by the lack of growth of many cancer cell lines in mice, the variability of growth kinetics and take rates from mouse to mouse. Drug efficacy studies are difficult due to the limited number of cell line-based models to test novel agents, the large sample sizes needed to power mouse in vivo studies, and the small tumor size and lack of ability to perform serial sampling of tumor and blood for pharmacodynamic/pharmacokinetic studies.
These challenges also occur in patient derived xenograft (PDX) models which are based on the transfer of primary tumors directly from the patient into an immunodeficient mouse, in which take rates are even lower and growth rates slower to obtain sufficient numbers of tumors for drug efficacy studies. PDX models have shown promise as clinical diagnostics to determine if a particular therapeutic regimen will be efficacious in a specific patient. However, PDX models are not routinely used because mouse hosts of PDX models can suffer from long latency periods after engraftment and variable engraftment rates (also referred to as “take rates”). Tumor graft latency, measured as the time between implantation and the development of a progressively growing xenograft tumor can range from two to twelve months (Siolas et al. Cancer Research 2013). In mice, the engraftment phase and expansion phase are often too long for the efficacy study to take place before the treatment of the patient must occur.
There is therefore a pressing need for models that provide improved take rates as well as growth rates for drug efficacy studies.
Disclosed herein are methods of screening a drug for treating a tumor comprising (a) administering the drug to a Severe Combined Immune Deficiency (SCID) rat having a xenograft tumor; wherein the SCID rat is a knockout rat comprising one or more genetic mutations that result in substantially depleted B-cells, T-cells and NK-cells. The effect of the drug on the tumor may then be determined. The SCID rat containing the xenograft tumor may be an F1 phase or an F2 passage. Further, the SCID rat may have a percentage take rate at least 10 points higher than a corresponding SCID mouse. The SCID rats disclosed herein may also exhibit tumor growth rate at least 5%, or more, than a corresponding SCID mouse. In particular aspects, the methods may use a SCID rat containing a deletion IL2Rg and the Rag2 genes; for example, the Rag2 deletion may comprise, or consist of, SEQ ID NO: 1, and the IL2Rg gene deletion may comprise, or consist of, SEQ ID NO:2.
Methods for performing drug efficacy assays using patient derived xenografts are also provided. The methods involve introduce introducing a patient derived xenograft, such as an ovarian cancer or a non-small cell lung cancer (NSCLC) into a Severe Combined Immune Deficiency (SCID) rat having substantially depleted mature B-cells, T-cells and NK-cells, and administering a drug to the rat. The rat may be a P1 or P2 passage rat. In addition to the methods disclosed herein, a Severe Combined Immune Deficiency (SCID) rat having a xenograft tumor for use in performing drug efficacy assays is provided.
As used herein, the term “take rate” refers to the percentage of animals in which a xenograft is found to grow.
As used herein, the term “about” refers to a value plus or minus 10% of the indicated numerical value.
As used herein, “coding sequence” refers to a nucleic acid, for example DNA, which, when expressed, results in the production of that RNA, polypeptide, protein, or enzyme. Coding sequence for a protein encompass a start codon (usually ATG) and a stop codon. Regulatory sequences that are involved in controlling expression of the coding sequence are outside of the coding sequence but remain part of the gene.
As used herein “deletion,” in the context of mutation, means a type of mutation that involves the removal of genetic material, which may be one or more nucleotides in the gene, including either the coding sequence of the regulatory sequences. Deletion may result in reduced or eliminated expression of the protein. In other aspects, the protein may be expressed but may have an altered sequence such that the protein no longer functions.
As used herein, “genetically modified” means a gene altered from its native state (e.g., by insertion mutation, deletion mutation), or that a gene product is altered from its natural state, using recombinant DNA techniques.
As used herein, “drug,” as used in the context of for example, drug efficacy or screening assays, encompasses both pharmaceutical-type drugs and biologic-type drugs such as antibodies.
As used herein, “knock-out” means a mutation in a gene of an animal, typically a rat, that reduces the biological activity of the polypeptide normally encoded by the gene by at least 80% compared to the unaltered gene. The mutation may be, for example, an insertion or a deletion resulting in frameshift mutation or missense mutation. Typically, the mutation is a deletion.
As used herein, the term “SCID” (Severe Combined Immune Deficiency) in the context of an animal refers to an animal having genetic mutations that result in depleted or substantially depleted B-cells, T-cells and NK cells. The animals typically are knock out animals with the result that that the proteins are not expressed at all, are expressed at such a low level that the protein does not support the normal biological function of the protein, or that the expressed protein is mutated with respect to the wild-type protein such that it does not support the normal biological function of the protein. In particular aspects, the SCID animal is a knock out rat with a mutation in both the Rag2 and Il2rg genes. In other aspects, the animal is a knock out animal with a mutation in both the Prkdc and Il2rg genes.
As used herein, a “corresponding” animal is an animal having the same functional differences, relative to the wild-type animal. For example, a SCID knock out mouse having mutated Prkdc and Il2rg genes may be a corresponding animal for a SCID rat having mutated Prkdc and Il2rg and may also be a corresponding animal for a SCID rat having mutated Rag2 and Il2rg genes. In each cases, the mutations in a corresponding animal results in a phenotype that is substantially identical to that in the animal from the other species.
As used herein, the term “substantially” refers to circumstance which is almost complete. For example, if a particular cell type is substantially depleted, only a residual amount of that type remains and is unable to support normal cell function. For example, if a cell type is substantially depleted, the amount of depletion is, compared to a normal circumstance, (e.g. wild type animal lacking identified mutations) decreased by at least 80%, at least 90%, at least 95%, or at least 99%, unless otherwise specified.
Disclosed herein are compositions and methods for producing transgenic rat models having improved engraftment of xenografts. The transgenic rat models are SCID rats. In particular, the rats are depleted or substantially depleted with respect to B-cells, T-cell, and NK cells. In particular, disclosed herein for the first time is a homozygous Rag2, Il2rg double knockout SCID rat model (Rag2−/−, IL2rg−/−) on the Sprague-Dawley strain as a competent host for human cancer cell lines and efficacy studies as well as human PBMCs for immune system humanization. In contrast to the corresponding mouse, the Rag2−/−, IL2rg−/− rat is a valuable in vivo human tumor model with the potential for immuno-oncology studies.
The SCID rat hosts of PDX models with improved engraftment efficiency and faster growth kinetics (engraftment and expansion phases from
Methods of Preparing the Rat KO
The SCID rats may be prepared using a variety of gene editing techniques; for example, they may be prepared using zinc-finger nucleases, CRISPR/Cas, TALEN, such as XTN™. See, for example, U.S. Pat. Nos. 8,993,233, 8,795,965, 8,771,945, 8,889,356, 8,865,406, 8,999,641, 8,945,839, 8,932,814, 9,902,971, each of which is incorporated by reference for all purpose, and in particular for methods of gene editing. In certain aspects, the CRISPR/Cas system can employ a Cas9 nuclease, which in some instances, is codon-optimized for the desired cell type in which it is to be expressed. The system further employs a fused crRNA-tracrRNA construct that functions with the codon-optimized Cas9. This single RNA is often referred to as a guide RNA or gRNA. Within a gRNA, the crRNA portion is identified as a target sequence for the given recognition site and the tracrRNA is often referred to as the ‘scaffold’. This system has been shown to function in a variety of eukaryotic and prokaryotic cells. Briefly, a short DNA fragment containing the target sequence is inserted into a guide RNA expression plasmid. The gRNA expression plasmid comprises the target sequence (in some embodiments around 20 nucleotides), a form of the tracrRNA sequence (the scaffold) as well as a suitable promoter that is active in the cell and necessary elements for proper processing in eukaryotic cells. The system may rely on complementary oligonucleotides that are annealed to form a double stranded DNA and then cloned into the gRNA expression plasmid. The gRNA expression cassette and the Cas9 expression cassette are then introduced into the cell. See, for example, Mali P et al. (2013) Science 2013 Feb. 15; 339 (6121):823-6; Jinek M et al. Science 2012 Aug. 17; 337(6096):816-21; Hwang W Y et al. Nat Biotechnol 2013 March; 31(3):227-9; Jiang W et al. Nat Biotechnol 2013 March; 31(3):233-9; and, Cong L et al. Science 2013 Feb. 15; 339(6121):819-23, each of which is herein incorporated by reference.
After preparing of a single knock out, the founders may be mated to produce a double knockout SCID rat. In certain aspects, the IL2rg and/or RAG2 genes contain a mutation that eliminates expression or reduces expression such that the animal does not produce an amount of protein adequate to carry out normal function. In certain aspects, the mutation is a deletion. In preferred aspects, the SCID rat is homozygous for a Rag2 deletion and an IL2rg deletion (i.e., Rag2−/−, IL2rg−/−). In certain embodiments, the sequence deleted from the Rag2 gene comprises SEQ ID NO:1 and the sequence deleted from the IL2rg sequence comprises SEQ ID NO:2. In other aspects, the sequence deleted from the Rag2 gene consists of SEQ ID NO:1 and the sequence deleted from the IL2rg sequence consists of SEQ ID NO:2. In particular aspects, no other genes are mutated compared to the wild-type animal.
Analysis of whole blood demonstrated that while a wild-type rat has about 37.4% CD4+, about 36.6% CD8+, and about 3.5% CD4+/CD8+ in the circulating lymphocytes. In contrast, the SCID rat has depleted or substantially depleted mature CD4+, CD8+ and NK cells compared to the wild-type. Thus, in preferred aspects, the CD4+ cells are present at about 1.0% to about 2.0%, for example about 1.5%, compared to about 37.4% percent in a wild-type animal. Thus, in particular aspects, depletion of CD4+ T cells may be at least 80%, at least 85% or at least 90%. In certain aspects, depletion of CD8+ T cells may be at least 80%, at least 85% or at least 90%.
In other aspects, CD8+ cells are present at about 4-6%, for example about 5%, compared to about 36.6% in the wild-type animal. The portion of CD4+/CD8+ cells in the SCID rat double knock out may be about 0.5% to about 1.5%; for example, about 1.2%, in contrast to about 3.5% in the while type animal. In yet other preferred aspects, the ILR2g and Rag2 KO rat is devoid of mature B cells in the spleen and/or in the circulation, using cell surface markers CD45 or IgM for detection.
In particular aspects, the NK cells are also substantially depleted or depleted. For example, the circulating NK cells, as measured by CD161 antibody, may be less than 1.0%; for example, about 0.5% or about 0.1% to about 0.9%, as a percentage of PBMCs. In contrast, a wild-type rat has circulating NK cells about 10%. Thus, in particular aspects, depletion of circulating NK cells is at least 85%, at least 90% or at least 95%.
In preferred aspects, the rat is a Sprague Dawley rat. In other aspects, the rat is a Long Evans rat, a Wistar Kyoto rat, a Fischer 344 rat or a Brown Norway rat.
SCID Rat Xenograft Model
The Rag2−/−, IL2rg−/−SCID rat is particularly useful as a model because of the unexpectedly superior growth rates compared to similar mouse models. In the mouse 4 to 8 months are required for establishment of a PDX model with enough animals for a preclinical study. In contrast, the Rag2−/−,IL2rg−/− shows xenograft tumor growth rates that makes the rat ready for screening drugs at a much earlier timepoint than would have been expected, which provides an excellent commercial advantage. Thus, in contrast to the rat models disclosed herein, the corresponding mouse does not effectively grow xenograft tumors. The contrast is illustrated in
In contrast, the SCID rats have better take rates than the equivalent SCID mouse for cancer xenografts. In particular aspects, the rat take rate is about 30%, about 40%, about 50%, about 70%, about 80%, about 90%, or about 100%, whereas the corresponding mouse take rate is lower by about 10 points, about 20 points, about 30 points, about 40 points, about 50 points, about 60 points, about 70 points, or about 80 points. Thus, for example, where a rat xenograft has a take rate of about 90%, the corresponding mouse xenograft take rate may be about 70 points lower; i.e. about 20%. The take rate is measured at a suitable time-point; for example, at 10 days post-implantation.
In other aspects, the SCID rats have better growth kinetics for the cancer xenografts. For example, the tumor growth rate in the rat is about 5%, about 10%, about 20%, about 50%, about 75%, about 100%, 200%, 500%, 1000% or more faster than a corresponding SCID mouse. Percentage tumor volume is calculated as: % Group Mean Change=((X−X0)/X0)*100 where X=current mean, X0=Initial mean. In particular examples, the % tumor volume group mean for IL2rg;Rag2 KO rat and corresponding SCID mouse over comparable time points was for HCT116 1,971% and 315% respectively, and for VCaP 2,133% and 530%, respectively.
The enhanced tumor growth kinetics results in a rat having a xenograft tumor with a tumor volume in a range of about 20,000 to about 40,000 mm3, or about 1000 mm3 to about 10,000 mm3 or about 100 mm3 to 1,000 mm3 or about 10,000 mm3 to 20,000 mm3 In particular examples, the range is about 700 to about 25,000 mm3 for VCaP about 500 to about 10,000 mm3 for H358, about 200 to about 6,000 mm3 for HCT116 and about 2,000 to about 20,000 mm3 for OCI-AML2. While the tumor volume varies with the xenograft, the permissive growth environment of the rat insures dramatically improved results compared to a corresponding mouse.
Compared to the corresponding SCID mouse xenograft model this means that time to establish the SCID rat xenograft model—for example, for use in drug efficacy testing—is reduced. For example, the time to establish a SCID rat xenograft model may be about 1 month less than the mouse, about 2 months less than the mouse, about 3 months less than the mouse, about 4 months less than the mouse, about 5 months less than the mouse, or about 6 months less than the corresponding SCID mouse xenograft model.
Thus, in particular aspects, the rat xenograft models disclosed herein are ready for performing assays (e.g. a drug efficacy assay) at the passage 1 (P1) stage or P2 stage. In other aspects, where a xenograft is particularly challenging, the rat model may not be ripe for assays until P3, P4, P5 or later stages. The P0 animal is the animal that first receives the xenograft. The P1 animal receives the xenograft from the P0 animal, and so on. In each case, however, the rat model is available earlier than the corresponding mouse is ready. For example, the rat xenograft model is ready at least 1 passage earlier, at least 2 passages earlier, or at least 3 passages earlier, than the corresponding mouse.
When the xenograft is from a patient (i.e., a PDX), the biopsy tissue may be removed and preserved in typical cell culture medium and is then cut into portions which are then introduced into the SCID rat. In certain aspects, the biopsy portion introduced is substantially cube-like and has dimensions of 2 mm along each side.
Moreover, the Rag2−/−, IL2rg−/−SCID rat is superior to the single Rag2−/− rat (referred to herein as SDR). In an experiment using the OCI-AML2 cell line as a xenograft, the Rag2−/−, IL2rg−/− SCID rat showed a 100% take rate, whereas the Rag2−/− rat showed a 0% take rate.
Xenografts
A variety of xenografts may be used for growth in the Rag2−/−, IL2rg−/− SCID rat. In some aspects, the xenograft is produced using established cancer cell lines. In other aspects, an implant may be prepared from a tumor sample taken from a patient during biopsy. The ability to grow such biopsy tissue (See
Advantageously, the NSCLC PDX model exhibits particular phenotypes useful for drug testing and for research. The size of the NSCLC PDX tumor can exceed 28 mm, with the volume over 32000 mm3 and we have found that under circumstances where the tumor length is greater than 25 mm, the core of the xenograft contains necrotic tissue. At later stages of development, the inner core of the tumor will be necrotic tissue, surrounded by a shell of quiescent cells (which often proliferate subsequent to therapy), and an outermost layer of live, proliferating cells. While most anti-cancer therapies focus on the proliferating cells, the rat models disclosed provide opportunities to assess the role of quiescent cells and the necrotic core in tumor pathology and in response to drug treatment, as well as the proliferating cells. The ability of the rat model to support tumor growth that accurately mimics naturally-occurring tumors reinforces the excellent utility of the rat model.
In particular aspects, the xenograft may be grown using cancer cell line; typically, a human cell line. The cell line may be from a variety of cancer types; for example, the cell line may be a breast cancer cell line, a prostate cancer cell line, a melanoma cell line, a colon cancer cell line, a lung cancer cell line, a lymphoma cell line, a pancreatic cancer cell line, an endometrial cancer cell line, a thyroid cancer cell line, an ovarian cancer cell line, or a bladder cancer cell line.
In some aspects, the breast cancer cell line is selected from the group consisting of MCF7, BT-20, MDA-MB-231, MDA-MB-453, and BT474. In some aspects, the colon cancer cell line is selected from SW-620, HCT116, SW-480, HT-29, and CT-26. In some aspects, the prostate cancer cell line is selected from the group consisting of VCaP, LNCaP, PC-3, 22Rv1, and DU-145. In some aspects, the leukemia cell line is selected from the group consisting of Jurkat, MV4-11, HL-60, THP-1, and REH. In some aspects, the lung cancer cell line is selected from the group consisting of A549, Calu-6, H358, Calu-3, and KYSE-30. In some aspects, the bladder cancer cell line is selected from group consisting of 786-0, A498, SW 780, and A498. In some aspects, the ovarian cancer is selected from the group consisting of SK-0V-3, OVCAR-3, OVCAR-5, and A2780. In some aspects, the brain cancer cell line is selected from the group consisting of U251 and U87-MG. The hepatocellular cancer cell line HepG2 may be used. The cell line may be the pancreatic cell line MiaPaCa-2 or PANC-1. The melanoma cell line may be A375. In other aspects, the FaDu cell line, derived from a squamous cell carcinoma, may be used. In preferred aspects, the cell line is selected from the group consisting of VCaP, H358, and HCT-116. In particular aspects, the cell line is OCI-AML2.
The cell lines are grown using conventional cell culture approaches and then injected into the animals subcutaneously. Typically, the cells are injected with a component that mimics extracellular matrix. Suitable extracellular matrix mimics may contain one or more of laminin, entactin/nidogen, collagen and heparan sulfate proteoglycans, and also growth factors like TGF-beta and EGF. Commercially available options include Cultrex® BME3 (Trevigen® #3632-001-02), Geltrex® (Gibco™), and Matrigel® (Corning®).
The number of cells introduced into the rat to form the xenograft may vary. For example, the number of cells may be in a range from about 1×106 to about 10×106, about 1×106 to about 5×106, or about 5×106 to about 10×106. Counting of cell numbers may be performed by methods known in the art.
In certain aspects, the xenograft introduces a human immune system into the Rag2−/−, IL2rg−/− SCID rat. Thus, in certain aspects, the xenograft comprises, or consists of, peripheral blood mononuclear cells (PBMCs). Previous attempts to use mice with similar genetic defects failed because the animals developed signs of GvHD at a timepoint that was sufficiently early such that the model was not useful. In contrast, however, implanting PBMCs into a rat SCID model did not result in problematic onset of GvHD symptoms. Thus, in particular aspects, disclosed herein are SCID rats having a humanized immune system having delayed onset of GvHd symptoms. In particular aspects, GvHD does not appear until after 6 weeks post-implantation, after 7 weeks post-implantation, after 8 weeks post-implantation, after 9 weeks post-implantation, after 10 weeks post-implantation, or after 11 weeks post-implantation. Thus, in contrast to mice where GvHD onset is about 4-5 weeks, the disclosed Rag2−/−, IL2rg−/− SCID rat model provides greater utility for studies requiring analysis of the human immune system. In certain other aspects, the xenograft used to produce a humanized immune system may comprise or consist of human hematopoietic stems cells (HSC), including, for example CD34+ HSC cells.
The xenograft SCID rats containing humanized immune systems may be used in methods to assess various responses including tumor-immune system interactions, tumor immune system escape, and the therapeutic effect of immune system modulation on tumor growth.
In particular aspects, a xenograft tumor may be removed from the rat and introduced into a second animal type, where the second animal type differs from the first. In some aspects, the second animal type is a rat with a different genetic background. In other aspects, the second animal type is not a rat. For example, the second animal type may be a mouse, a dog, a rabbit, a hamster, a macaque, or a chimpanzee. In particular aspects, the mouse may be a knock-out mouse. In some cases, the knock-out mouse may be a comparable knock-out mouse; in other cases, the knock-out mouse may have different genes knocked out. Advantageously, the excellent xenograft growth in the rat provides a substantial amount of tumor tissue that can divided amongst multiple animals for performing assays. Such an approach offers particular economic value as performing assays in the mouse requires less materiel and mice are cheaper to use as a model generally. In particular aspects, the xenograft type is one that grows well when directly introduced into the second animal type; in other aspects, the xenograft type is one that does not grow well when directly introduced into the second animal but shows improved growth kinetics in the second animal type; for example, in a mouse following growth in the SCID rat.
Drug Efficacy Assays
The SCID rat knock-out models containing xenografts disclosed herein are particularly useful for drug efficacy studies. The route of administration of the drug may be subcutaneous, intraperitoneal, intravascular (intravenous and intra-arterial), intramuscular, topical, intradermal, oral, mucosal, or ocular. In particular aspects, administration is by tail vein injection.
A variety of pharmaceutical and biologic anti-cancer drugs may be tested. For example, the drug may be a PARP inhibitor such as Talazoparib (BMN-673), Veliparib, Olaparib, Rucaparib (AG014699, PF-01367338), Veliparib (ABT-888).CEP 9722[30], E7016, BGB-290. In certain aspects, the drug may be an antisense oligonucleotide, a microRNA, or an RNAi. In other aspects the drug may be designed as a cancer-specific drug. For example, enzalutamide is a suitable prostate cancer drug. In yet other aspects, the drug is a biologic such as an antibody, a CAR-T cell, or another cell-based therapy.
Efficacy of the drug may be determined by any suitable means. For example, in certain assays, the response to the drug may be measured related to impact on tumor growth compared to control. In other aspects, particular molecules produced by the tumor or cells expressing particular genes may also be monitored (for example, by PCR or by FACS analysis); for example, where the xenograft is derived from a prostate cancer, either by biopsy or by using cancer cell line such as VCap, the amount of Prostate Serum Antigen (PSA) produced by the xenograft may be monitored.
It is a particular advantage of the rat xenograft models that the excellent take rate and rapid tumor growth allow drug studies to be initiated soon after xenograft implantation. In particular aspects, the rat is ready for drug studies (e.g. a screen or assay) at 1, 2, 3, 4, 5, or 6 months post-implantation of the xenograft.
In other aspects, the xenograft tumor may be removed from the rat and used to perform assays in vitro. Advantageously, the high take rate and growth kinetics mean that, compared to an equivalent mouse, about 10-fold more cellular tissue is available for such assays. Thus, in particular aspects, the methods disclosed herein include growing a xenograft in a SCID rat model, harvesting cells of the xenograft, and using the harvested cells in an assay. Because the xenografts grow well, cell harvesting may take place about 1, 2, 3, 4, 5, or 6 months post-implantation of the xenograft.
The SCID rat models having a humanized human system may be used in a variety of ways. For example, they may be used to determine the impact of cell therapies such as chimeric antigen receptor (CAR)-T cells. In other aspects, they may be used to assess blockade of checkpoint proteins; for example, they may use to test efficacy of drugs that modulate the activity of programmed cell death protein 1 (PD-1), PD-L1, or CTLA-4. For example, new antibodies against checkpoint inhibitors may be assayed.
Generation of Rag2- and Rag2/IL2RG-Knockout Sprague Dawley Rats
For the single knockout, the Rag2 locus was targeted using XTN™ technology in spermatogonial stem cells (SSCs). Pooled SSCs were transplanted into DAZL-deficient sterile males and mated with wild-type Sprague Dawley rats. DNA was isolated from offspring and a male with a 27 bp deletion was detected.
For the double-knockout, the Rag2 and Il2rg loci were targeted using CRISPR. CRISPR based targeted nuclease reagents targeting the Rag2 and Il2rg genes were microinjected into Sprague Dawley embryos at the 2-cell stage. A total of 314 embryos were injected, of which 187 were successfully transferred into pseudopregnant surrogates. 32 animals were born of which, 9 animals carried at least one mutated allele verified by targeted sequence analysis. These founders were interbred to create the Rag2.I2rg double knockout animal (Rag2−/−, IL2rg−/−), which contains an 8 bp homozygous deletion in the Rag2 gene (atatggcc; SEQ ID NO:1) and 16 bp homozygous deletion (gagaatctaggctcat; SEQ ID NO:2) in the Il2rg gene.
FACS analysis of immune cells: To detect T, B, and NK cells, flow cytometric analysis was performed on splenocytes and thymocytes. Cells were stained with fluorophore-labeled antibodies at a final concentration of 25 μg/ml in 20 μl volume for 20 minutes. Antibodies used were Goat anti-rat IgM-APC (Stem Cell Technologies #10215), PE Mouse anti-rat IgM (BD #553888), FITC Mouse anti-rat CD45R (BD #561876), APC Mouse anti-rat CD45R (BD #554881), FITC Mouse Anti-Rat CD8b (BD #554973), PE Mouse anti-rat CD8b (BD #554857) APC Mouse Anti-Rat CD4 (BD #550057), FITC Mouse Anti-Rat CD161a (BD #561781), APC Mouse anti-rat CD161a (BD #555009).
Transplantation of human cancer cell lines: 1 million cells (U87MG human glioblastoma) or 1, 5, or 10 million cells (H358 human non-small cell lung cancer cells) were mixed with Geltrex® 1:1 and transplanted subcutaneously in the hind flank. Tumors were measured three times weekly and recorded in StudyLog to determine tumor growth kinetics. Animals were euthanized before the tumors reached humane endpoints.
Immunohistochemistry for human proteins: Tumors were excised and fixed in 10% NBF. Standard Sum sections were collected and human cells were visualized by staining with an antibody that recognizes a protein found in all human mitochondria (mouse anti-human mitochondria antibody, clone 113-1; EMD Millipore #1273) at 1:250.
Improved Human Non-Small Cell Lung Cancer (NSCLC) Tumor Engraftment and Kinetics in Rag2 KO Rats
The Rag2 knockout, demonstrated improved tumor growth kinetics and engraftment rate for H358 xenografts. A KRAS mutant non-small cell lung cancer (NSCLC) cell line H358 was implanted into Rag2 KO rats subcutaneously. 1, 5, or 10 million cells (H358 human non-small cell lung cancer cells) were mixed with Geltrex® 1:1 and transplanted subcutaneously in the hind flank. Tumors were measured three times weekly and recorded in StudyLog to determine tumor growth kinetics.
The tumor growth was faster and more consistent when compared to NSG and Nude Mice (
Improved Human Prostate Cancer Tumor Engraftment and Kinetics in Rag2−/−, IL2rg−/− Rats
In a study for the evaluation of growth kinetics for VCaP human prostate tumor xenograft model using Nod SCID and ICR SCID, the tumor engraftment rate and growth kinetics were poor. In both models 3 of 5 mice showed tumor engraftment but not until at least day 28 and 32 for NOD and ICR SCID mice respectively (
However, when the same VCap human prostate tumor xenograft model was implanted on both flanks of ILR2g and Rag2 KO rats, we achieved tumor growth within less than 14 days. For transplantation, 10×106 VCaP cells for each animal were resuspended in 250 μL sterile 1×PBS (Gibco #14190-144). Immediately prior to injection, 250 μl 10 mg/ml Cultrex BME3 (Trevigen #3632-001-02) was added to the cell suspension for a final Cultrex concentration of 5 mg/ml. The cell/Cultrex suspension was injected subcutaneously into the hindflank. Tumor diameter was measured using digital calipers (Fisher #14-648-17) 3 times a week. Tumor volume was calculated as (L×W2)/2, where width and length were measured at the longest edges
Enhanced Growth with HCT-116 Xenograft Model in the ILR2g and Rag2 KO SCID Rat Vs. NSG Mouse.
We also assessed the ability of the HCT-116 xenograft to form tumor xenografts.
For transplantation, 2×106 HCT-116 cells for each animal (NSG mice and ILR2g and Rag2 KO rats) were resuspended in 250 μl sterile 1×PBS (Gibco #14190-144). Immediately prior to injection, 250 μl 10 mg/ml Cultrex BME3 (Trevigen #3632-001-02) was added to the cell suspension for a final Cultrex concentration of 5 mg/ml. The cell/Cultrex suspension was injected subcutaneously into the hindflank. Tumor diameter was measured using digital calipers (Fisher #14-648-17) 3 times a week. Tumor volume was calculated as (L×W2)/2, where width and length were measured at the longest edges.
Humanization of Immune System
We prepared Rag2;Il2rg KO rats as described above. We then injected 20×106 viable human PBMCs in 500 μl PBS into the tail vein of 3 male and 3 female rats at 8-10 weeks of age. Peripheral blood was analyzed for the presence of human CD45+, CD3+, CD4+, CD8+ and CD20+ cells at 3, 7, 14, 28, 54, and 70 days post-injection.
After tail vein injection of 50 million human PBMCs, we were able to detect human CD45+ in the circulating blood of 66% of recipient rats. As expected, the majority of these CD45+ cells were also human CD3+. By 28 days post-transplant, nearly 100% of hCD45+ cells detected in the peripheral blood were CD3+. Interestingly, a subset of successfully engrafted rats had a small percentage of human CD45+ cells that were CD3−, which we later determined were human CD20+, a marker of B cells.
In the SCID mice transplanted with human PBMCs, there are no reports of circulating human B cells, though the spleens of these mice contain a significant population of human CD20 cells. In contrast to the mouse, we determined that the ILR2g and Rag2 KO rats contained circulating B cells based on the presence of circulating CD20+ cells.
We also determined that the ILR2g and Rag2 KO SCID rats displayed graft versus host disease (GvHD), marked by rapid body weight loss, loss of body condition, and lymphocyte infiltration in peripheral tissues. While GvHD is a hallmark of successful engraftment of human PBMCs, due to the presence of mature human T cells which eventually attack the recipient, it is notable that the ILR2g and Rag2 KO rats with successful engraftment did not exhibit symptoms until 8 weeks post-transplantation or later, a later onset compared to PBMC-engrafted mice.
Lung Cancer Primary Tumor Implant Xenograft
We compared the ability of primary tumors from patients to grow in NSG mice and ILR2g and Rag2 KO rats. NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) were obtained as described above. ILR2g and Rag2 KO rats were prepared as described in Example 1. We obtained tissue to use as a primary cell implant from a patient diagnosed as having NSCLC. We obtained the biopsy sample in media: DMEM (Gibco #11965092), 10% FBS (Atlanta Biologicals #S12452), 1% Penicillin/Streptomycin (Gibco #15070063), 1% Amphotericin B (Gibco #15290026). The biopsy tissue was rinsed in PBS. The biopsy was cut into approximately cube-like structures with 2 mm side length for implantation. These pieces were kept in media on ice until ready to transplant. Prior to implantation the tissue piece is rinsed in sterile PBS. A sterile forcep was used to place it into an autoclaved trocar and the tissue (in the trocar) was kept in the dish of PBS to prevent it from drying out.
A small (1 mm) incision was made just below the left shoulder blade with sterile scissors or a 16 G needle. Tenting the skin at the incision using sterile forceps, the trocar was placed into the incision. The trocar was gently pushed and guided through the subcutaneous (SQ) space until the tip reached the left dorsal hind flank. The trocar plunger was then inserted to push the biopsy piece into the SQ space.
After introducing the tissue into ILR2g and Rag2 KO rats and into NSG mice we compared the ability of those two models to grow the primary implant.
We obtained essentially identically superior results with an ovarian primary tumor. Compare
All patents, patent applications, and published scientific papers are incorporated by reference for all purposes.
This application claims priority to U.S. Prov. No. 62/479,857 filed Mar. 31, 2017, the disclosure of which is hereby incorporated by reference for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2018/025368 | 3/30/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62479857 | Mar 2017 | US |
