FOLATE RECEPTOR ALPHA -TARGETING BI-SPECIFIC T CELL ENGAGERS (BiTEs) AND USES THEREOF
20240408202 ยท 2024-12-12
Inventors
Cpc classification
C07K14/705
CHEMISTRY; METALLURGY
A61K39/39
HUMAN NECESSITIES
C07K16/2809
CHEMISTRY; METALLURGY
H10F30/2255
ELECTRICITY
A61K40/11
HUMAN NECESSITIES
A61K40/4202
HUMAN NECESSITIES
C12N2740/10043
CHEMISTRY; METALLURGY
C07K16/28
CHEMISTRY; METALLURGY
A61K2239/59
HUMAN NECESSITIES
C12N15/86
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
A61K39/00
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
C07K16/28
CHEMISTRY; METALLURGY
Abstract
Provided are modified cells and methods for their use in treating cancer. The cells are modified to express and secrete a Bi-specific T cell engager (BiTE) that includes a segment that specifically binds to human Folate Receptor alpha (FR) and a segment that that specifically binds to human CD3, such as CD3e. The modified cells can be T cells. Methods for producing the modified cells are also provided.
Claims
1. A modified cell that is modified to express and secrete a Bi-specific T cell engager (BiTE), wherein the BiTE includes segment that specifically binds to human Folate Receptor alpha (FR) and a segment that that specifically binds to human CD3, wherein the human CD3 is optionally CD3e.
2. The modified cell of claim 1, wherein the modified cell is a modified human T cell.
3. The modified human T cell of claim 2, wherein the segment that specifically binds to the human Folate Receptor alpha (FR) comprises amino SEQ ID NO:6, and wherein the segment that specifically binds to human CD3 specifically binds to human CD3e and comprises the sequence of SEQ ID NO:5.
4. The modified human T cell of claim 2, wherein the BiTE comprises amino acids 1-531 of SEQ ID NO:10 and wherein the BiTE is secreted from the modified human T cell.
5. A method comprising introducing into an individual in need of treatment for cancer modified human cells of claim 1.
6. The method of claim 5, wherein the modified human cells are T cells.
7. The method of claim 6, wherein in the modified human T cells the segment of the Bi-specific T cell engager (BiTE) that specifically binds to the human Folate Receptor alpha (FR) comprises amino SEQ ID NO:6, and wherein the segment that specifically binds to human CD3 specifically binds to human CD3e and comprises the sequence of SEQ ID NO:5.
8. The method of claim 7, wherein the BiTE comprises amino acids 1-531 of SEQ ID NO:10.
9. The method of claim 8, wherein the cancer comprises a solid tumor.
10. The method of claim 9, wherein the solid tumor comprises ovarian cancer cells.
11. The method of claim 10, wherein the modified human T cells are administered a single time and have therapeutic efficacy.
12. The method of claim 10, wherein the modified human T cells are administered in consecutive doses, and wherein the consecutive doses are optionally separated by a period of time comprising more than 24 hours, and wherein said doses have therapeutic efficiency.
13. A method of making modified human cells that express and secrete a Bi-specific T cell engager (BiTE) as in claim 1, the method comprising conditioning the cells in a medium that comprises interleukin (IL) IL-2, IL-7, IL-15, or a combination thereof, and introducing into the cells a polynucleotide encoding the BiTE, wherein the conditioning is performed prior to introduction of the polynucleotide, concurrent with introduction of the polynucleotide, or after introduction of the polynucleotide.
14. The method of claim 13, wherein the modified human cells are T cells.
15. Modified human cells made according to the method of claim 13.
16. A method comprising administering to an individual in need of treatment for cancer modified human cells of claim 13.
17. The method of claim 16, wherein the individual is in need of treatment for ovarian cancer.
18. A polynucleotide that encodes a Bi-specific T cell engager (BiTE) as in claim 1.
19. The polynucleotide of claim 18, wherein the polynucleotide is present in a viral expression vector.
20. The polynucleotide of claim 19, wherein the viral vector is a retroviral vector.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] Where reference to a figure number includes a letter the letter means a panel on the numbered figure.
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DETAILED DESCRIPTION
[0024] Unless defined otherwise herein, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.
[0025] The following abbreviations are used in this disclosure: [0026] ACT: Adoptive T cell Transfer [0027] ANOVA: Analysis of variance [0028] BiTEs: Bispecific T cell engagers [0029] BiTE-T cells: BiTE-secreting T cells [0030] CAR: Chimeric Antigen Receptor [0031] cDMEM: Complete DMEM (media) [0032] CONT-ENG: Control engager [0033] CTV: CellTrace Violet [0034] cRPMI: Complete RPMI (media) [0035] E:T: Effector to target ratio [0036] FACs: Fluorescence activated cell sorting [0037] FBS: Fetal bovine serum [0038] FR: Folate receptor alpha [0039] FR-B T cells: Folate receptor alpha bispecific T cell engager-secreting T cells (mouse) [0040] FR-Bh T cells: Folate receptor alpha bispecific T cell engager-secreting T cells (human) [0041] GFP: Green fluorescent protein [0042] GM-CSF: Granulocyte-macrophage colony-stimulating factor [0043] Gyn Onc: Gynecologic oncology [0044] hFR: Human Folate receptor alpha [0045] hrs: Hours [0046] IFN-: Interferon gamma [0047] IHC: Immunohistochemistry [0048] IL-2, 5, 6, 7, 13, 15: Interleukin 2, 5, 6, 7, 13, 15 [0049] IP: Intraperitoneal [0050] IU: International unit [0051] Luc/GFP: Luciferase-Green fluorescent protein fusion protein [0052] MIP-1/: Macrophage inflammatory protein 1 alpha and beta [0053] MIRV: mirvetuximab soravtansine [0054] ml: Milliliter [0055] mM: Millimolar [0056] OC: Ovarian cancer [0057] PBS: Phosphate buffered saline [0058] PDX: Patient-derived xenograft [0059] RNAseq: RNA sequencing [0060] scFv: Single chain variable fragment [0061] SQ: Subcutaneous [0062] TALs: Tumor-associated lymphocytes [0063] TBI: Total Body Irradiation [0064] TCR: T cell receptor [0065] TILs: Tumor infiltrating T cells [0066] TME: Tumor microenvironment [0067] Tregs: Regulatory T cells [0068] TSCM: Stem cell memory T cells [0069] UTD: Untransduced
[0070] Every numerical range given throughout this specification includes its upper and lower values, as well as every narrower numerical range that falls within it, as if such narrower numerical ranges were all expressly written herein.
[0071] The disclosure includes all polynucleotide and amino acid sequences described herein. Amino acids of all protein sequences and all polynucleotide sequences encoding them are also included, including but not limited to sequences included by way of sequence alignments. Sequences of from 80.00%-99.99% identical to any sequence (amino acids and nucleotide sequences) of this disclosure are included. The disclosure includes all amino acid and polynucleotide sequences that are identified herein by way of a database entry as the sequences exist in the database as of the effective filing date of this application.
[0072] The disclosure includes all compositions, results, and method steps alone and in combination, and described herein and as depicted in the Figures.
[0073] As discussed above, the disclosure provides binding partners provided as bispecific T cell engagers (BiTEs). Thus, the binding partners are in certain examples are multivalent. In embodiments, leukocytes, including but not necessarily limited to T cells, express a BiTE.
[0074] In one approach the present disclosure combines BiTE-based technologies and the therapeutic approach of targeting FR to develop a novel ACT approach for OC and other cancers that utilizes engineered FR-targeted BiTE-T cells (referred to from time to time herein as FR-Bh T cells for human T cells, and FR-B T cells for mouse T cells). As demonstrated in the Examples and Figures of this disclosure, FR-Bh T cells were highly effective against both FR+OC patient samples and in immunocompetent preclinical tumor models. Moreover, mechanistic studies revealed that improved therapeutic efficacy was accompanied by preferential accumulation of less differentiated stem-like FR-B T cells in the extratumoral peritoneal OC TME over solid tumor lesions. This indicates that FR-B T cells in remote locations can promote tumor destruction in OC (by secreting BiTEs and engaging endogenous T cells) without a requirement for direct accumulation in solid tumors. The disclosure is therefore expected to be suitable for use as an ACT therapy used to treat solid tumors, including but not necessarily limited to OC, where limited tumor reactivity from endogenous T cells can create therapeutic challenges.
[0075] In embodiments, binding partners of this disclosure may comprise linking sequences. Suitable amino acid linkers may be mainly composed of relatively small, neutral amino acids, such as glycine, serine, and alanine, and can include multiple copies of a sequence enriched in glycine and serine. In specific and non-limiting embodiments, the linker comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 amino acids. In an example, the linker may be the glycine-serine-alanine linker G.sub.4SA.sub.3 (SEQ ID NO:18) or a glycine-serine linker (G.sub.4S).sub.4 (SEQ ID NO:19) linker. Representative examples of linking sequences are provided below in the described sequences.
[0076] In embodiments, such as for proteins that are produced as a fusion protein, a peptide linker may be used, and may comprise a cleavable or non-cleavable linker. In embodiments, the peptide linker comprises any self-cleaving signal. In embodiments, the self-cleaving signal may be present in the same open reading frame (ORF) as the ORF that encodes the binding partner. A self-cleaving amino acid sequence is typically about 18-22 amino acids long. Any suitable sequence can be used, non-limiting examples of which include: T2A, P2A, E2A, and F2A, the sequences of which are known in the art.
[0077] In embodiments, a binding partner may include a secretion signal. The present disclosure therefore provides modified cells, such as T cells, that secrete a described binding partner. For secretion, any suitable secretion signal can be used and many are known in the art. In non-limiting embodiments, the secretion signal comprises MALPVTALLLPLALLLHA (SEQ ID NO:1), METDTLLLWVLLLWVPGSTG (SEQ ID NO:2), or MGWSCIILFLVATATGVHSD (SEQ ID NO:3) or GEAAAKEAAAKEAAAK (SEQ ID NO:8). Representative examples of proteins produced and secreted by modified cells according to this disclosure are described further below. For amino acid sequences of this disclosure that include amino acids that comprise purification or protein production tags, including but not limited to HIS tags, the disclosure includes the proviso that the sequences of any described tag may be excluded from the claimed amino acid sequences. Further, where linker sequences are identified, any suitable linker sequence may be substituted for the described sequence. Linker sequences and/or purification tag sequences may be excluded from sequence similarity values described herein. Any binding partner described herein may be fully or partially humanized.
[0078] For therapeutic approaches, in certain embodiments, a described binding partner may be delivered as mRNA or DNA polynucleotides that encode the binding partner. It is considered that administering a DNA or RNA encoding any binding partner described herein is also a method of delivering such binding partners to an individual or to one or more cells, provided the DNA is transcribed and the mRNA is translated, and/or the RNA itself is delivered and translated. Methods of delivering DNA and RNAs encoding proteins are known in the art and can be adapted to deliver the binding partners, given the benefit of the present disclosure. In embodiments, one or more expression vectors are used and comprise viral vectors. Thus, in embodiments, a viral expression vector is used. Viral expression vectors may be used as naked polynucleotides, or may comprises any of viral particles, including but not limited to defective interfering particles or other replication defective viral constructs, and virus-like particles. In embodiments, the expression vector comprises a modified viral polynucleotide, such as from an adenovirus, a herpesvirus, or a retroviral vector. In embodiments, the retroviral vector is adapted from a murine Moloney leukemia virus (MLV) or a lentiviral vector may be used, such as a lentiviral vector adapted from human immunodeficiency virus type 1 (HIV-1). Representative demonstrations of the disclosure are provided and use mouse stem cell virus (MSCV)-based retroviral vector to produce retrovirus in Platinum-E (PLAT-E) virus packaging cells for mouse T cell transduction or PG13 virus packaging cells for human T cell transduction.
[0079] In alternative embodiments, a recombinant adeno-associated virus (AAV) vector may be used. In certain embodiments, the expression vector is a self-complementary adeno-associated virus (scAAV).
[0080] In embodiments, cells modified according to this disclosure include mature T cells, or their progenitor cells such hematopoietic stem cells or any other time of T cell progenitor cells. The disclosure includes progeny of progenitor cells. Thus, in embodiments, cells that are modified to express any binding partner described herein include but are not necessarily limited CD4+ T cells, CD8+ T cells, Natural Killer T cells, T cells, and cells that are progenitors of T cells, such as hematopoietic stem cells or other lymphoid progenitor cells, immature thymocytes (double-negative CD4-CD8-) cells, or double-positive thymocytes (CD4+CD8+). In embodiments, the progenitor cells comprise markers, such as CD34, CD117 (c-kit) and CD90 (Thy-1). In embodiments, a population of human peripheral blood mononuclear cells are modified using the described polynucleotides.
[0081] In embodiments, a polynucleotide that encodes a described binding partner selectively hybridizes to a polynucleotide encoding at least one protein that is a component of a binding partner, including but not limited to a heavy chain CDR1, CDR2, and CDR3 of any described binding partner. In embodiments, the polynucleotide selectively hybridizes to a polynucleotide encoding a light chain CDR1, CDR2 and CDR3 of any described binding partner. In embodiments, the polynucleotide selectively hybridizes to a polynucleotide encoding CDR1, CDR2 and CDR3 of a heavy and light chain of any described binding partner.
[0082] Pharmaceutical formulations containing binding partners are included in the disclosure, and can be prepared by mixing them with one or more pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers include solvents, dispersion media, isotonic agents and the like. The carrier can be liquid, semi-solid, e.g. pastes, or solid carriers. Examples of carriers include water, saline solutions or other buffers (such as phosphate, citrate buffers), oil, alcohol, proteins (such as serum albumin, gelatin), carbohydrates (such as monosaccharides, disaccharides, and other carbohydrates including glucose, sucrose, trehalose, mannose, mannitol, sorbitol or dextrins), gel, lipids, liposomes, resins, porous matrices, binders, fillers, coatings, stabilizers, preservatives, liposomes, antioxidants, chelating agents such as EDTA; salt forming counter-ions such as sodium; non-ionic surfactants such as TWEEN, PLURONICS or polyethylene glycol (PEG), or combinations thereof.
[0083] In embodiments, an effective amount of T cells expressing a described binding partner is administered to an individual in need thereof. In embodiments, an effective amount is an amount that reduces one or more signs or symptoms of a disease and/or reduces the severity of the disease. An effective amount may also inhibit or prevent the onset of a disease or a disease relapse. A precise dosage can be selected by the individual physician in view of the patient to be treated. Dosage and administration can be adjusted to provide sufficient levels of binding partner to maintain the desired effect. Additional factors that may be taken into account include the severity and type of the disease state, age, weight and gender of the patient, desired duration of treatment, method of administration, time and frequency of administration, drug combination(s), reaction sensitivities, and/or tolerance/response to therapy. In embodiments an effective amount is an amount of modified T cells that express and secrete a binding partner and produces a therapeutic effect without of the modified T cells in a solid tumor that is 1-20% of the total T cells present in the solid tumor. In embodiment the engineered T cells are about 10% of the total T cells in a solid tumor (see, for example,
[0084] The described binding partners and T cells that express and secrete the binding partners can be administered directly or provided as pharmaceutical compositions and administered to an individual in need thereof using any suitable route, examples of which include intravenous, intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, oral, topical, or inhalation routes, depending on the particular condition being treated. Intra-tumor injections may also be used. The compositions may be administered parenterally or enterically. The compositions may be introduced as a single administration or as multiple administrations or may be introduced in a continuous manner over a period of time, non-limiting examples of which are demonstrated herein.
[0085] In embodiments, the described compositions are suitable for use in humans. The disclosure also includes the described constructs that are suitable for use in syngeneic immunocompetent mouse models.
[0086] In embodiments, the individual in need of a composition of this disclosure has been diagnosed with or is suspected of having cancer. In embodiments, the cancer is a solid or liquid tumor. In embodiments, the cancer is renal cell carcinoma, breast cancer, prostate cancer, pancreatic cancer, lung cancer, liver cancer, ovarian cancer, cervical cancer, colon cancer, esophageal cancer, glioma, glioblastoma, or another brain cancer, stomach cancer, bladder cancer, testicular cancer, head and neck cancer, melanoma or another skin cancer, any sarcoma, including but not limited to fibrosarcoma, angiosarcoma, adenocarcinoma, and rhabdomyosarcoma, and any blood cancer, including all types of leukemia, lymphoma, and myeloma.
[0087] In embodiments, administering a described binding partner, such as by way of administering T cells that are modified to secrete the described binding partner, exhibits an improved activity relative to a control. In an embodiment, the control comprises administration of a BiTE without using T cells that secrete the BiTE. In embodiment, the control comprises a BiTE that is secreted by a cell that is not a T cell.
[0088] The described T cells that express the described binding partners can be combined with any other therapeutic agent, non-limiting examples of which include conventional chemotherapeutic agents, and immune checkpoint inhibitors, the latter of which are known in the art, and target CTLA4, PD-1, or PD-L1. Thus, the disclosure includes combination therapy using one or more described binding partners and any of CTLA-4 inhibitors, PD-1 inhibitors and PD-L1 inhibitors. As non-limiting examples, anti-PD-1 agents include Pembrolizumab and Nivolumab. Anti-PD-L1 examples include Avelumab and Atezolizumab. An anti-CTLA-4 example is Ipilimumab. The binding partners may also be combined with any other form of adoptive immunotherapy. The modified T cells may be used in autologous or allogenic therapies.
[0089] The disclosure includes the described expression vectors that encode the BiTEs, and all methods of making T cells that are described herein and by way of the figures. In embodiments, the disclosure provides for obtaining T cells from an individual and subjecting the T cells to cytokine treatment to prepare the T cells for use as an adoptive immunotherapy, and modifying the T cells to express the described BiTEs. The T cells may be modified to express the described BiTEs before, during or after cytokine treatment, but the cytokine treatment is before perfusion into an individual. In embodiments, the cytokine treatment comprises repeated IL-15 treatments. In embodiments, the cytokine treatment comprises IL-2 +IL-7, or IL-2+IL-15. In embodiments, substituting IL-15 in place of IL-7 enhances properties of the T cells, such as expansion/persistence following infusion, a stem-like phenotype, and improved tumor control, as illustrated in the Figures.
[0090] As is known in the art, BiTEs incorporate single-chain variable fragments (scFvs) and are composed of a tumor-targeted scFv providing tumor antigen target specificity, linked in tandem to a T cell specific scFv, which provides T cell activation (typically an anti-CD3 scFv).
[0091] The disclosure provides non-limiting examples of embodiments that are illustrated in the Figures and described in the Examples below. Data produced and described in the accompanying figures showing production of modified cells and results from use of said cells that express and secrete the BiTEs were obtained using polynucleotides that encode the following sequences:
TABLE-US-00001 Anti-mouseCD3escFV Clone:145-2C11 Format:VH/VL StandardG4SLinker(bold) (SEQIDNO:4) EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRGLESVAY ITSSSINIKYADAVKGRFTVSRDNAKNLLFLQMNILKSEDTAMYYCARFD WDKNYWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLPASLGDRV TINCQASQDISNYLNWYQQKPGKAPKLLIYYTNKLADGVPSRFSGSGSGR DSSFTISSLESEDIGSYYCQQYYNYPWTFGPGTKLEIK Anti-humanCD3escFv Clone:UCHT1 Format:VL/VH StandardG4SLinker(bold) (SEQIDNO:5) MDIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIY YTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFA GGTKLEIKGGGGSGGGGSGGGGSGGGGSEVQLQQSGPELVKPGASMKISC KASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTV DKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS Anti-humanFRscFv Clone:MOv19 Format:VL/VH Non-standard(referredtoas212) Linker(bold) (SEQIDNO:6) DIELTQSPASLAVSLGQRAIISCKASQSVSFAGTSLMHWYHQKPGQQPKL LIYRASNLEAGVPTRFSGSGSKTDFTLNIHPVEEEDAATYYCQQSREYPY TFGGGTKLEIKGSTSGSGKSSEGKGQVQLQQSGAELVKPGASVKISCKAS GYSFTGYFMNWVKQSHGKSLEWIGRIHPYDGDTFYNQNFKDKATLTVDKS SNTAHMELLSLTSEDFAVYYCTRYDGSRAMDYWGQGTTVTVS BiTESequences MouseFRtargetedBiTE(mCD3escFvxhFR scFv);FR-B mCD3escFV=145-2C11Clone hFRscFV=Mov19Clone *ChimericBiTE=bindshumantargetantigenand mouseCD3foruseinsyngeneicimmunocompetent mousemodels *SignalSequence(secretionsignal)at Nterminusinitalics (SEQIDNO:8) GEAAAKEAAAKEAAAK linkersequence;Rigidandlonglinkersequence betweenscFvwastestedinparallelwithmultiple linkersofvaryinglength/flexibilityandwas foundtoresultinoptimalantigenbindingbyboth CD3eandFRtargetedscFv'sinthecurrent formatandresultedinoptimalinvivoactivity *Shortlinker+6xHisTagatCterminus (SEQIDNO:7) MNSGLQLVFFVLTLKGIQGEVQLVESGGGLVQPGKSLKLSCEASGFTFSG YGMHWVRQAPGRGLESVAYITSSSINIKYADAVKGRFTVSRDNAKNLLFL QMNILKSEDTAMYYCARFDWDKNYWGQGTMVTVSSGGGGSGGGGSGGGGS DIQMTQSPSSLPASLGDRVTINCQASQDISNYLNWYQQKPGKAPKLLIYY TNKLADGVPSRFSGSGSGRDSSFTISSLESEDIGSYYCQQYYNYPWTFGP GTKLEIKGEAAAKEAAAKEAAAKDIELTQSPASLAVSLGQRAIISCKASQ SVSFAGTSLMHWYHQKPGQQPKLLIYRASNLEAGVPTRFSGSGSKTDFTL NIHPVEEEDAATYYCQQSREYPYTFGGGTKLEIKGSTSGSGKSSEGKGQV QLQQSGAELVKPGASVKISCKASGYSFTGYFMNWVKQSHGKSLEWIGRIH PYDGDTFYNQNFKDKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGS RAMDYWGQGTTVTVSSGSGHHHHHH HumanFRtargetedBiTE(hCD3escFVx hFRscFv) hCD3escFv=UCHT1clone hFRascFv=Mov19clone *SignalSequenceatNterminusinitalics (SEQIDNO:8) GEAAAKEAAAKEAAAK; (SEQIDNO:9) G(EAAAK) 3linkersequence: SamelinkersequenceasusedinmouseFR targetedBiTE Shortlinker+6xHisTagatCterminus (SEQIDNO:10) MNSGLQLVFFVLTLKGIQGMDIQMTQTTSSLSASLGDRVTISCRASQDIR NYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQ EDIATYFCQQGNTLPWTFAGGTKLEIKGGGGSGGGGSGGGGSGGGGSEVQ LQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINP YKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYG DSDWYFDVWGQGTTLTVFSGEAAAKEAAAKEAAAKDIELTQSPASLAVSL GQRAIISCKASQSVSFAGTSLMHWYHQKPGQQPKLLIYRASNLEAGVPTR FSGSGSKTDFTLNIHPVEEEDAATYYCQQSREYPYTFGGGTKLEIKGSTS GSGKSSEGKGQVQLQQSGAELVKPGASVKISCKASGYSFTGYFMNWVKQS HGKSLEWIGRIHPYDGDTFYNQNFKDKATLTVDKSSNTAHMELLSLTSED FAVYYCTRYDGSRAMDYWGQGTTVTVSSGSGHHHHHH.Thedis- closureincludesuseofthisconstructwithamino acids1-531only.
[0092] For use in humans, in an embodiment a binding partner of this disclosure includes a sequence that is an anti-human CD3e scFv as shown in SEQ ID NO:5. In an embodiment a binding partner of this disclosure includes a sequence that is an anti-human FR scFv sequence as shown in SEQ ID NO:6. In an embodiment a binding partner of this disclosure includes both a sequence that is an anti-human CD3e sequence and a sequence that is an anti-human FR, such as the sequence shown in SEQ ID NO:10) wherein said sequence includes amino acids 1-531 only and therefore excludes the HIS tag.
Example 1
[0093] The constructs described above were analyzed as described in the following methods.
Cell Culture
[0094] SK-OV-6 (cervical), SK-OV-3 (ovarian), OV167 (ovarian), OVCAR8 (ovarian), OVCAR3 (ovarian), K562 (Leukemia), IE9-mp1 (ovarian), IE9-mp1-hFRa (ovarian), Pan02-hFRa (pancreatic) cancer cell lines were grown in complete RPMI (cRPMI) containing 10% FBS, 25 mM Hepes, 2 mM L-Glutamine, 100 IU/ml Pen/Strep, 1 mM Sodium Pyruvate, 1 Non-Essential Amino Acids, and 0.05 mM -Mercaptoethanol. 293T, PG13, and PLAT-E cell lines were grown in complete DMEM (cDMEM) containing 10% FBS and 100 IU/ml Pen/Strep. Cell lines were IMPACT tested and/or confirmed mycoplasma negative prior to use.
Generation of hFra-Expressing Cell Lines, BiTE Constructs and Retroviral Vectors
[0095] An aggressively growing and immunotherapy resistant IE9-mp1 variant recovered at disease relapse following immunotherapy.sup.29 was used to generate the human FR (hFRa)-expressing IE9-mp1-hFRa cell line using the Sleeping Beauty Transposon system as detailed in herein. As a second murine model, Pan02-hFRa cells was also produced. FR-Bh binds human FR via a scFv derived from the MOv19 antibody and human CD3 via a scFv derived from the UCHT1 antibody. FR-B binds human FR as above and mouse CD3 via a scFv derived from the 145-2C11 antibody. The design/construction of all engager sequences, production of retroviruses, and testing for engager binding are described below.
T Cell Activation and Transduction
[0096] Human or mouse T cells were activated using anti-CD3 and anti-CD28 antibodies (Bio X Cell) prior to retroviral transduction. Specific activation/culture conditions and retroviral transduction protocols is described herein.
In Vitro Co-Cultures
[0097] Human or mouse T cells were cultured for no less than 8 days post activation before assay set up. T cells were co-cultured with target cells at the indicated E:T ratios in cRPMI for 24 or 48 hrs. For serial stress test studies involving repeated and prolonged co-culture of mouse T cells with target cells, T cells were harvested, counted, and resuspended in fresh cRPMI+ cytokine support (IL-2+IL-7 or IL-2+IL-15 as indicated) at the start of each new 3-day co-culture period. Additional details are included herein.
OC Patient Samples and Targeting Using BiTE-T Cells
[0098] Cryopreserved OC patient ascites samples (Supplemental Table 1 (shown in
Preclinical Mouse Models and Therapeutic Delivery of T Cells
[0099] FR-Bh T cell evaluation in the SK-OV-6 human xenograft model is described in the supplemental methods. For studies using immunocompetent mice, 6-8-week-old female C57BL/6J mice were purchased from the Jackson Laboratory and housed in the Roswell Park Comparative Oncology Shared Resource (COSR). 510.sup.6IE9-mp1-hFRa cells (IP in 500 l PBS) or 210.sup.6 Pan02-hFRa (SQ in 100 ul PBS) were injected to establish tumors, with ACT commenced 5 days later. Mice received 8.3310.sup.5-310.sup.6 FR-B T cells or an equal number of unarmed control T cells (Luc/GFP transduced or mock transduced) delivered by loco-regional injection (IP or intratumoral delivery for SQ tumors), with timing/dosing as indicated. FR-B T cell accumulation in the blood, peritoneal TME, or solid tumors was assessed 5 days post ACT. Additional details related to in vivo studies, tissue collection, processing, and analysis have been included in the supplemental methods.
Antibodies and Flow Cytometry Staining/Analysis
[0100] Antibodies for flow cytometry were purchased from BioLegend or BD Biosciences and have been listed in Supplemental Table 2 (shown in
Statistical Analysis
[0101] Two-tailed, unpaired and paired t tests were used to compare data between two groups. One- and two-way Analysis of Variances (ANOVA) were used for data analysis of more than two groups and/or across multiple time points and a Tukey post-test was utilized to determine significant differences between groups. Survival data was compared using a Logrank test. Results were generated using GraphPad Prism software. Differences between means were considered significant at p<0.05: * p<0.05, ** p<0.01, *** p<0.001, **** p<00.0001.
[0102] The results in the following Examples were produced using the materials and methods described in Example 1.
Example 2
Human BiTE-Secreting T Cells have Specificity for FR+ Cancer Cells and Actively Target OC
[0103] To target FR+OC, we generated a FR-specific BiTE by linking a human CD3-specific scFv (UCHT1) and a MOV19-derived FR-specific scFv using optimized linker sequences (7
[0104] To test if FR-Bh T cells could target clinically-relevant OC, FR-Bh or CONT-ENG T cells were co-cultured at a T cell: tumor cell ratio of 4:1 with OC patient specimens (isolated from peritoneal ascites at the time of surgery, Suppl. Table 1, shown in
[0105] To gain insights into the breadth of inflammatory changes driven by FR-Bh T cell therapy against clinical OC specimens, we selected 4 OC patients that responded to FR-Bh T cells from the Fr.sup.Int and Fr.sup.hi cohorts and broadly analyzed immunological changes in co-cultures containing CONT-ENG T cells or FR-Bh T cells using the Isoplexis Human Adaptive Immune Codeplex Secretome Panel (
Example 3
FR-Bh T Cells Effectively Engage OC Patient T Cells Present in the Peritoneal Tumor Microenvironment
[0106] Based on these data, we reasoned that both FR-Bh T cells and endogenous patient T cells present in the OC TME may be actively engaged following delivery of FR-Bh T cells, thereby contributing to the BiTE-driven T cell response. To permit separate interrogation of BiTE-T cell versus host T cell activation in co-cultures containing OC patient specimens, exogenously added T cells (comprised of engineered FR-Bh/CONT-ENG-producing and bystander non-transduced T cells) were labeled with CellTrace Violet (CTV) prior to addition to co-cultures, permitting discrete assessment of transferred [CTV+; transduced (GFP+) and UTD bystander (GFP) T cells] and endogenous (CTV-GFP) T cells (
Example 4
Therapeutic Delivery of BiTE-Secreting T Cells Improves Tumor Control and Survival in an Immunocompetent OC Model
[0107] Given that FR-Bh T cells engaged/activated endogenous T cells in OC patient samples, we next tested the therapeutic delivery of FR-directed BiTE-T cells in an immunocompetent OC mouse model. To do so, an aggressively growing and immunotherapy-resistant variant of the IE9-mp1 OC cell line.sup.29 was engineered to stably express human FR (IE9-mp1-hFR) and a chimeric BiTE specific for human FR and mouse CD3 was generated (hereafter referred to as FR-B) (
[0108] To evaluate FR-B T cells therapeutically, IE9-mp1-hFRa tumor-bearing mice were treated with FR-B or unarmed control T cells (either UTD or T cells engineered to express a Luciferase-GFP fusion protein; Luc/GFP) and monitored for tumor progression and survival (
Example 5
Stem-Like FR-B T Cells can be Produced Through Cytokine Preconditioning and Improve Antitumor Immunity Following ACT
[0109] Based on our in vivo findings, we evaluated whether strategies to improve FR-B T cell persistence following infusion would improve therapeutic efficacy. As IL-15 stimulation has been shown to promote a less-differentiated stem cell memory (T.sub.SCM) phenotype, increase mitochondrial metabolic fitness, and improve T cell persistence following infusion of CAR-T cells.sup.33, and can enhance the activity of BiTE-T cells.sup.34, we tested whether IL-15 preconditioning prior to ACT would impact FR-B T cell efficacy and response durability against OC. As FR-B T cells were produced in the presence of IL-2 and IL-7 (FR-B 2/7) in prior experiments, we directly compared this approach to FR-B T cells produced using IL-2 and IL-15 stimulation (FR-B 2/15). FR-B 2/7 and FR-B 2/15 T cells were generated with similar efficiency by retroviral transduction (
Example 6
IL-2/IL-15 Preconditioning Improves FR-B T Cell Persistence in the Extratumoral OC Peritoneal TME
[0110] To better understand the improved antitumor effects of FR-B 2/15 T cells, we compared the tissue localization of FR-B 2/7 and FR-B 2/15 T cells following infusion. FR-B CD4+ T cells demonstrated limited accumulation in the blood, peritoneal TME (TALs), as well as solid tumor lesions (TILs), with no clear differences between 2/7 and 2/15 preconditioned FR-B CD4+ T cells (
[0111] In contrast to the blood and solid OC tumors, the frequency of FR-B 2/15 CD8+ TALs in the peritoneal cavity was elevated more than 3-fold compared to FR-B 2/7 TALs (
[0112] Transcriptional profiling of flow cytometry-sorted CD8+FR-B 2/15 and FR-B 2/7 TALs isolated 5 days post ACT suggested limited differences in effector function or expression of checkpoint pathways between the transferred T cells (
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Discussion of Examples Above
[0160] The present disclosure demonstrates robust activity of FR-B(h) T cells that was accompanied by engagement of endogenous T cells in the OC TME, thereby overcoming limited endogenous immunoreactivity or local tumor immunosuppression. Delivery of T cells by IP injection has been shown to result in accumulation of infused T cells in solid tumors in the peritoneal cavity.sup.32, which is consistent with the present data shown for FR-B T cells. However, FR-B T cells comprised only a small fraction of the TILs found in solid OC and the improved therapeutic effects of FR-B 2/15 over FR-B 2/7 T cell therapy correlated with differences in FR-B T cell accumulation outside of solid tumors (
[0161] A recent report demonstrated that tumor-specific CD8+ T cells that infiltrate and remain in tumors for at least 72 hrs upregulate checkpoint receptors and can rapidly develop an exhausted phenotype.sup.37, emphasizing that i) limiting BiTE-T cell infiltration into tumors may be beneficial for prolonging BiTE-T cell activity and ii) the activation of endogenous T cells by secreted BiTEs indicates the presence of newly infiltrating (and not yet exhausted) tumor-specific T cells or activation of bystander T cells that remain functional in the TME. Additionally, the instant data indicate that effector-like FR-B 2/7 CD8+ TALs increase fatty acid/lipid metabolism within the OC TME, metabolic reprogramming that has been associated with PD-1 signaling.sup.38 and suggesting FR-B T cells can also be impacted by inhibitory cues in the broader peritoneal OC TME that may promote early T cell clearance. Moreover, a recent report demonstrated that CD39-expressing CD8+ T cells can directly suppress the antitumor activity of tumor-specific T cells.sup.39 suggests the predominantly CD39+FR-B 2/7 FR-B TALs may actually limit tumor attack within the OC TME.
[0162] The disclosure includes use of the FR-B T cells to localize to other sites in the peritoneal space, including tumor-draining lymph nodes or the spleen. A small frequency of FR-B T cells was also observed in circulation, supporting loco-regional delivery of FR-B T cells leading to antitumor immunity at distant metastatic sites.
[0163] In addition to elevated IFN- levels, we also noted increased production of Th2-associated cytokines (in particular IL-5) in response to FR-Bh T cells in OC patient samples, suggesting BiTE-driven activity of diverse T cell subsets. Engaging multiple T cell subsets, whether CD8+ T cells or differentially polarized CD4+ T cells (which can include Tregs).sup.40 may impact therapeutic response, particularly as T cells present in the TME at the time of ACT may exist in multiple heterogenous states.sup.41. We also observed a reduction in IL-6 in OC patient samples treated with FR-Bh T cells, which contrasts with CAR-T cell therapy where elevated IL-6 has been associated with increasing severity of cytokine release syndrome.sup.42.
[0164] The disclosure includes use of the described BiTE-T cells with a co-stimulatory signal and/or cytokines. It is considered that because soluble BiTEs effectively combine with blockade of checkpoint receptors including PD-1 and CTLA-4.sup.17, it is considered likely that the described FR-B(h) T cells will synergize with checkpoint blockade for treating OC.
[0165] The disclosure includes multi-arming T cells, for example with CARs and the described BiTEs to target multiple tumor antigens which may overcome tumor heterogeneity and/or elicit immune attack on multiple target cell subsets.
[0166] The presently provided results demonstrate the potent effects of FR-B(h) T cells for ACT in OC, which can effectively redirect endogenous T cells to amplify antitumor immunity. The results also reveal a unique attribute of FR-B T cells in OC to persist and direct antitumor activity from solid tumor-adjacent or extratumoral locations in the peritoneal TME, which may have distinct mechanistic advantages for enhancing response durability following ACT.
Example 7
This Example Provides Supplemental Methods in Respect of the Prior Examples.
Generation of hFra-Expressing Cell Lines, BiTE Constructs and Retroviral Constructs
[0167] Human FR (hFRa) was PCR amplified from cDNA of SK-MEL-37 melanoma cell line using the following primers
TABLE-US-00002 FWD: (SEQIDNO:11) TGTCGTGAAAACTACCCCGCGGCCGCCACCATGGCTCAGCGGATGACAAC ACA and REV: (SEQIDNO:12) TTCGTGGCTCCGGAGCCACTGCTGAGCAGCCACAGCAGCATT.
The hFRa gene was genetically fused to the monomeric enhanced GFP (eGFP) reporter via SGSG-linker and a P2A translational skipping sequence and inserted into the pT2-EF 30 sleeping beauty transposon plasmid.sup.1 using NEBuilder HIFI DNA assembly (New England Biolabs), with sequences confirmed by Sanger Sequencing at the Roswell Park Genomics Shared Resource. The pT2-EF-hFR-GFP vector was co-electroporated with the CMV(CAT)T7-SB100 transposase vector (Addgene plasmid #34879; RRID:Addgene_34879) into IE9-mp1 and Pan02 cell lines using the Nucleofector 4D Instrument. Electroporated cells were cultured for 10-14 days prior to FACs sorting of GFP.sup.hi cells using a BD FACSAria II cell sorter. Sorted cells were confirmed to express hFRa by flow cytometry. A hFR-specific scFv with murine immunoglobulin kappa light chain was designed by fusing MOv19 kappa chain (Sequence ID: X99994.1) and heavy chain (Sequence ID: X99993.1) sequences via a 212 polypeptide-containing linker (GSTSGSGKSSEGKG (SEQ ID NO:13)) and was synthesized by Integrated DNA technologies gBlock. FRB is a chimeric BiTE that binds human FR and mouse CD3e (via a previously described scFv derived from the 145-2C11 monoclonal antibody.sup.2. These scFvs are linked by a rigid and long G(EAAAK SEQ ID NO:9)x3 linker sequence that resulted in optimal antigen binding and in vivo FR-B activity compared to a panel of tested linkers (data not shown). The BiTE leader sequence.sup.2 and 145-2C11 derived scFv sequence were codon-optimized and synthesized by gBlock (Integrated DNA technologies), with the FR-B sequence designed to contain a 6 His Tag at the C terminus. The FR-B sequence was genetically fused to the monomeric enhanced GFP (eGFP) reporter via a SGSG (SEQ ID NO:9)-linker and P2A translational skipping sequence to allow monitoring of transduction efficiency (
T Cell Activation and Transduction
[0168] For human studies, peripheral blood mononuclear cells (PBMC) were isolated from the whole blood of healthy donors and received under an approved Biospecimen and Data Research (BDR) protocol. PMBC were activated using precoated plate-bound anti-human CD3 antibody (OKT-3, 5 g/ml prepared in PBS, Bio X Cell) for 48 hrs in cRPMI containing anti-human CD28 antibody (9.3, 2 g/ml, Bio X Cell), human IL-2 (50 U/ml, Peprotech), and human IL-7 (10 ng/ml, BioLegend). For murine studies, splenocytes were harvested from female C57BL/6J or T-Lux 3 mice, subjected to RBC lysis using ACK lysis buffer, and activated using precoated plate-bound anti-mouse CD3 (145-2C11, 5 g/ml prepared in PBS, Bio X cell) for 48-72 hrs in cRPMI containing anti-mouse CD28 antibody (37.51, 2 g/ml, Bio X Cell), human IL-2 (50 U/ml, Peprotech), and either mouse IL-7 (10 ng/ml, BioLegend) or mouse IL-15 (10 ng/ml BioLegend). Following activation, T cells were harvested, counted, and loaded onto Retronectin (Takara)-coated non-tissue culture treated plates preloaded with retrovirus by spinning cleared cell supernatants from high-titer retrovirus-producing PG13 (human) or PLAT-E (mouse) cells at 3000 rpm for 1 hr at 32 C. (2 cycles of retrovirus preloading completed prior to T cell loading). T cell transduction was conducted on two consecutive days, followed by at least 24 hr T cell expansion prior to assessment of T cell transduction efficiency (based on GFP+ cells, gated using GFP mock transduced T cells) by flow cytometry. Following activation, T cells were maintained in cRPMI containing cytokine support (IL-2+IL-7 or IL-15), which was replaced every 2-3 days.
Antibodies and Flow Cytometry Staining/Analysis
[0169] For mouse samples, Fc blocking was performed by using an anti-CD16/CD32 antibody (2.4G2, Bio X Cell, 15 min at 4 C.) to inhibit non-specific antibody binding prior to surface staining. For intracellular staining, The BD Transcription Factor Buffer Set (BD Biosciences) was used according to the manufacturer's suggested protocol. In cases where fixation/permeabilization were performed, intracellular staining for GFP was additionally included to permit interrogation of FR-B T cells based on GFP in fixed cells. For human samples, blocking was performed using human FcR Block (Miltenyi Biotech) for 10 min at 4 C., followed by staining (30 mins, 4 C.) using antibodies prepared in either FACs Buffer or BD Horizon Brilliant Staining Buffer. For human studies involving addition of engineered T cells to OC patient specimens, CONT-ENG or FR-Bh T cells were harvested and pre-labeled with CellTrace Violet (Thermo Fisher) according to the recommended protocol prior to addition. For all direct ex vivo mouse studies and for studies using human OC specimens, Zombie UV or Zombie Near-IR fixable viability staining (BioLegend) was performed to ensure interrogation of only viable cells. Stained Samples were collected using BD LSR II or Fortessa Flow cytometers and downstream data analyzed using FlowJo V10 software (BD Biosciences).
In Vitro Co-Culture Studies
[0170] To assess target cell killing, T cells were gently washed from cultures using cold PBS and target cells were enumerated by counting a minimum of 4 randomly selected regions of interest (ROI's)/well using the Cytation 5 instrument (Biotek) or quantified using the CellTiter-Glo 2.0 Cell Viability Assay (Promega) to determine % target cell killing compared to control wells containing target cells alone. T cell activation (FR-B, Luc/GFP, or Mock transduced T cells) was assessed after 24 hr co-culture with IE9-mp1-hFRa target cells using CD69 surface staining and flow cytometry. Culture supernatants from co-cultures were collected, spun down to remove debris, aliquoted and stored at 80 C. prior to analysis and were assessed for IFN- production using either the human or mouse IFN- ELISA MAX Deluxe set (BioLegend) according to the manufacturer's suggested protocol.
Transwell Assay to Assess BiTE-Mediated Bystander T Cell Activity
[0171] 210.sup.5 untransduced (UTD) human T cells were plated with (210.sup.4) SK-OV-6 cells (E:T=10:1) in 24 well tissue culture plates in 800 l cRPMI. Next, 0.4 m PETMembrane 24 well transwell inserts (Greiner Bio-One) were placed in the wells and 10.sup.6 human CONT-ENG or FR-Bh T cells were added to the transwell in 200 l cRPMI. CONT-ENG or FR-Bh T cells added directly to the SK-OV-6 target cells (no transwell) were used as negative and positive controls, respectively. Co-cultures were plated in technical duplicate or triplicate and incubated for 48 hrs in the presence of human IL-2 (50 U/ml), at which point T cells were gently washed from the lower chamber and target cells harvested and viable cells counted. Culture supernatants were collected and analyzed for human IFN- production by ELISA as described.
OC Patient Samples and Targeting Using FR-Bh T Cells
[0172] Patient samples were assessed for the frequency of tumor cells (CD45-EpCAM+) that were FR+ by flow cytometry. Following 48 hr co-culture, culture supernatants were collected for downstream analysis and cells collected for flow cytometry as detailed. To allow enumeration of FR+ and FRtumor cells following co-culture, CountBright Absolute Counting Beads (Thermo Fisher) were added prior to Flow cytometry analysis. Culture supernatants were spun down to remove debris, aliquoted and stored at 80 C. prior to analysis and were assessed for IFN- production using the human IFN- ELISA MAX Deluxe set (BioLegend) according to the manufacturer's suggested protocol. Additionally, 4 responding patient culture supernatants were selected to assess broad immunological effects between OC patient samples containing either CONT-ENG or FR-Bh T cells using the Isoplexis Human Adaptive Immune Codeplex Secretome to measure 22 inflammatory parameters using the Isoplexis Isolight system according to the manufacturer's recommended protocol.
Preclinical Mouse Models and Therapeutic Delivery of FR-B T Cells
[0173] For evaluation of FR-Bh T cell therapeutic activity using a human xenograft model, 310.sup.6 SK-OV-6 cells (prepared in 100 l PBS) were implanted subcutaneously in the flanks of female 6-8-week-old female NSG mice bred in the Roswell Park Laboratory Animal Shared Resource (LASR). Tumor volumes were calculated as 0.5(LengthWidth.sup.2) and when tumors reached 150 mm.sup.3, mice were stratified into groups to remain untreated or receive 310.sup.6 FR-Bh or Cont-ENG T cells (delivered as split dose between IV and intratumoral routes, prepared in 200 l PBS). Mice received 3 daily doses of 210.sup.4 U of IL-2 (IP injection, 200 l PBS) beginning on the day of T cell infusion. Changes in tumor volume were determined twice/week for the duration of study. In studies involving depletion of endogenous lymphocytes in immunocompetent mice, mice were treated with 5 Gy Total body irradiation immediately prior to tumor implantation and lymphodepletion confirmed by flow cytometry prior to adoptive T cell transfer. IP tumor progression was monitored based on increased abdominal distension (measured as changes in circumference) due to accumulation of peritoneal ascites, which closely correlates with solid tumor growth in this model.sup.4 5, with mice considered endpoint and euthanized when abdominal circumference reached 10 cm (or at earlier measurements if mice developed decreasing health status due to peritoneal disease progression). For subcutaneously implanted Pan02-hFR, tumor volume was calculated at 0.5(LengthWidth.sup.2), with mice considered endpoint and euthanized when tumor dimensions exceed 10 mm in both directions. All performed experiments and procedures were reviewed and approved by the Roswell Park IACUC prior to conducting experiments.
Monitoring of FR-B T Cell Responses in Treated Mice
[0174] Blood was collected by retro-orbital blood draw, peritoneal lavage collected following IP injection of PBS, and solid tumors excised from the omental region of animals. RBC lysis was performed on blood and peritoneal wash samples using ACK lysis buffer and solid tumors were processed using the gentleMACs Dissociator (Miltenyi Biotec), followed by passage through 70 m cell strainers. Samples were subsequently stained for flow cytometry analysis as outlined.
Metabolomic Assessment of FR-B T Cells Using Seahorse
[0175] FR-B 2/7 or FR-B 2/15 T cells were generated for comparison of metabolic function using the Mitochondrial Stress Test conducted using the Seahorse XFe96 Analyzer. Briefly, the Mitochondrial Stress Test was performed in XF DMEM Base Media with no Phenol red containing 10 mM glucose, 1 mM sodium pyruvate, and 2 mM L-glutamine and the following inhibitors were added at the following final concentrations: Oligomycin (2 M), Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) (1.0 M), Rotenone/Antimycin A (0.5 M each). Prepared T cells were plated on cell-tak (Corning) coated Seahorse XF96 cell culture microplates at a density of 2.510.sup.5 cells per well and in replicates of 8 wells per cell type. The assay plates were allowed to rest at RT for 45 mins and then spun for 5 minutes at 1,000 rpm. The plates were then incubated at 37 C. without CO2 for 10 mins prior to performing the assay on the Seahorse XFe96 Analyzer. Assay was set up and ran according to manufacturer's recommended method and post run data was obtained through Seahorse Wave Desktop Software, with additional statistical analysis completed using GraphPad Prism.
FACs Sorting of CD8+FR-B TALs, RNA Sequencing, and Transcriptomic Analysis
[0176] FR-B 2/7 or FR-B 2/15 T cells (8.410.sup.5/mouse, prepared in 200 l PBS) were adoptively transferred by IP injection into IE9-mp1-hFRa tumor-bearing mice and peritoneal washes collected from mice 5 days later following IP injection of 5 ml PBS. Collected cells were washed and immediately stained for cell viability (Zombie UV, prepared in PBS) followed by Fc blocking and surface phenotyping using antibodies prepared in BD FACs Pre-Sort Buffer (BD Biosciences). Live CD45+CD11b.sup.CD19.sup.CD4.sup. (Gated out using a PE-Cy7 Dump Channel) CD8+ GFP+FR-B TALs were sorted using a BD FACSAria II Cell Sorter and collected directly into PCR tubes containing 2 l of Takara Plain Sorting Solution with a target collection of 500 cells/sample. Sufficient cell input (359-500 cells) was achieved for downstream analysis using 5 unique biological samples (n=2 for FR-B 2/7 CD8+ TALs, n=3 for FR-B 2/15 CD8+ TALs). Sorted cells were kept on ice and immediately brought to the Roswell Park Genomics Shared Resource for further processing and RNAseq analysis. RNAseq analysis of sorted CD8+FR-B TALs was conducted using the Takara Bio USA, Inc. SMART-Seq v4 PLUS Kit. Final libraries were sequenced on an Illumina NovaSeq 6000 using 2100 sequencing and an average of 50 million paired reads/sample were generated. Following sequencing, samples were passed through Illumina bcl2fastq v2.20 to generate fastq files for downstream analysis. Bioinformatics pre-processing and quality control (QC) steps were carried out by the Roswell Park Bioinformatics Shared Resource, using an established pipeline following commonly adopted practices for RNA-seq data analysis. Raw reads that passed the Illumina RTA quality filter were demultiplexed and pre-processed using FastQC for sequencing base quality control. Raw reads that passed the Illumina RTA quality filter were demultiplexed and pre-processed using FastQC for sequencing base quality control. Reads were then mapped to the mouse reference genome (GRCm39) and reference transcriptome GENCODE (vM28) using STAR.sup.6. Raw feature counts were normalized and differential expression analysis was carried out using DESeq2.sup.7. Differential expression rank order was used for subsequent gene set enrichment analysis (GSEA).sup.8, performed using the cluster profile package in R. Gene sets queried included the Hallmark, Canonical pathways, and GO Biological Processes Ontology collections available through the Molecular Signatures Database (MSigDB).sup.9.
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