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WO2025054284A1 - Refinement of car constructs via barcoded screening - Google Patents

Refinement of car constructs via barcoded screening Download PDF

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WO2025054284A1
WO2025054284A1 PCT/US2024/045332 US2024045332W WO2025054284A1 WO 2025054284 A1 WO2025054284 A1 WO 2025054284A1 US 2024045332 W US2024045332 W US 2024045332W WO 2025054284 A1 WO2025054284 A1 WO 2025054284A1
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car
domain
antigen
cells
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Xavier RIOS
Leonid METELITSA
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Baylor College of Medicine
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C12N15/1027—Mutagenizing nucleic acids by DNA shuffling, e.g. RSR, STEP, RPR
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    • C12N15/09—Recombinant DNA-technology
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1065—Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C07K2319/00—Fusion polypeptide
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    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
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    • C12N2510/00—Genetically modified cells

Definitions

  • the present disclosure relates generally to the fields of protein engineering development. More particularly, it concerns methods to develop new chimeric antigen receptor (CAR)-based cancer immunotherapies through combinatorial screening and methods of use thereof.
  • CAR chimeric antigen receptor
  • CAR-based cancer immunotherapy has proven successful in treating hematologic malignancies, leading to complete remission for many patients with refractory hematologic malignancies.
  • CAR-based immunotherapies use synthetic receptors that redirect immune effector cells to target cancer cells based on recognition of extracellular antigens.
  • receptors consist of several essential components: 1) an extracellular antigenrecognition domain, typically a single-chain variable fragment (scFv); 2) a hinge region (or connector) linking the extracellular domain to the transmembrane domain; 3) a single-pass transmembrane domain; 4) one or two cytoplasmic costimulatory domains (second- or third- generation CARs, respectively) that enhance T cell activation by providing “signal two;” and 5) a cytoplasmic domain containing immunoreceptor tyrosine-based activation motifs (ITAMs), usually but not exclusively from CD3( ⁇ , that provides “signal one” and initiates the signaling cascade that leads to T cell activation.
  • ITAMs immunoreceptor tyrosine-based activation motifs
  • the present disclosure concerns methods and compositions related to constructing libraries of CAR constructs and the methods of their use.
  • An exemplary embodiment of the disclosure is a method of producing CAR constructs comprising the steps of: receiving a set of uniquely barcoded antigen-recognition domains comprising one or more unique antigen-recognition domains; receiving a set of uniquely barcoded connector domains comprising a plurality of unique connector domains; receiving a set of uniquely barcoded transmembrane domains comprising one or more unique transmembrane domains; receiving a set of uniquely barcoded costimulatory domains comprising a plurality of unique costimulatory domains; receiving a set of uniquely barcoded intracellular signaling domains comprising one or more unique intracellular signaling domains; and, ligating together the set of uniquely barcoded antigen-recognition domains, the set of uniquely barcoded connector domains, the set of uniquely barcoded transmembrane domains, the set of uniquely barcoded costimulatory domains, the set of uniquely barcoded costimulatory domains, such that each resulting
  • Embodiments of the method additionally comprise one or more of the following steps: the step of transfecting the CAR constructs into immune cells; subjecting the immune cells to antigen stimulation; testing the immune cells for proliferation, surface expression, relative expansion, antitumor activity, antivirus activity, antibacterial activity, antifungal activity, removal of cellular pathogenic state or a combination thereof; selecting the immune cells with the high antitumor activity, antivirus activity, or antibacterial activity; identifying the CAR construct based on the barcode; generating treatment immune cells comprising the CAR domains identified in the CAR construct; and, administering the treatment immune cells to a patient in need thereof.
  • the immune cells are T cells, NK cells, NKT cells, gamma-delta T-cells, Macrophages, neutrophils, and iPSC-derived effectors, or a combination thereof.
  • the CARs are delivered to immune cells in vivo via viruses or mRNA vaccines.
  • one of the one or more antigen recognition domains binds to CD 19, EBNA, CD123, HER2, CA-125, TRAIL/DR4, CD20, CD70, CD38, CD123, CLL1, carcinoembryonic antigen, alphafetoprotein, CD56, AKT, Her3, epithelial tumor antigen, CD319 (CS1), R0R1, folate binding protein, HIV-1 envelope glycoprotein gpl20, HIV-1 envelope glycoprotein gp41, CD5, CD23, CD30, HERV-K, IL-l lRalpha, kappa chain, lambda chain, CSPG4, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD70, TROP-2, CD99, p53, mutated p53, Ras, mutated ras, c-Myc, cytoplasmic serine/threonine kinases (e.g., A-R
  • one of the one or more unique antigen recognition domains from antibodies such as CD19 FMC63, GD2 14G2a, or a Designed Ankyrin Repeat Protein.
  • one of the plurality of unique connector domains comprises all or a portion of the hinge domains of CD28, CD8a, CD3( ⁇ , CD8b, CD4, 41bb, IgG4, IgGl, or IgG2.
  • one of the plurality of unique connector domains comprises SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO:5.
  • one the one or more unique transmembrane domains comprises all or a portion of the transmembrane domain CD28, CD8ot, DAP 10, CD3z, CD8a, CD28, CD8b, 41bb, CD40, CD4, CD3e, CD3g, or CD3d.
  • one of the one or more unique transmembrane domains comprises SEQ ID NO: 6.
  • one of the plurality of unique costimulatory domains comprises all or part of wildtype or mutant signaling domains from one or more of CD28, 41BB or 4-1BB (CD137), ICOS, CD27, CD40, 0X40 (CD134), DAP12, or Myd88.
  • one of the plurality of unique costimulatory domains comprises SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 .
  • one or more intracellular signaling domains comprises the activating domains from CD3( ⁇ , DAP10, DAP12, 2B4, CD3g, CD3d, CD3e, CD79a, CD79b, or FcRgamma.
  • one of the one or more unique intracellular signaling domains comprises ZAP70, SLP76, PLCyl, or LAT.
  • one or more of the intracellular signaling domains compromise Src family kinases suck as Lek.
  • one of the one or more intracellular signaling domains comprises SEQ ID NO: 12 or SEQ ID NO: 13.
  • the connector domain, the transmembrane domain, the costimulatory domain, the intracellular signaling domain undergo Type Ils digestion.
  • the ligation can be blunt end ligation.
  • the resulting CAR construct does not comprise any spacers or unnatural amino acid sequences between any of the domains.
  • Another exemplary embodiment of the disclosure is a library of barcoded CAR constructs comprising: a plurality of CAR constructs comprising one or more antigen-recognition domains, one connector domain, one transmembrane domain, zero, one or more costimulatory domains, and zero, one or more intracellular signaling domain and a unique barcode comprising, in order, an antigen-recognition domain identifying barcode, a connector domain identifying barcode, a transmembrane domain identifying barcode, one or more costimulatory domain identifying barcodes, and an intracellular signaling domain identifying barcode.
  • one of the one or more antigen recognition domain binds to CD 19, EBNA, CD 123, HER2, CA- 125, TRAIL/DR4, CD20, CD70, CD38, CD123, CLL1, carcinoembryonic antigen, alphafetoprotein, CD56, AKT, Her3, epithelial tumor antigen, CD319 (CS1), ROR1, folate binding protein, HIV-1 envelope glycoprotein gpl20, HIV-1 envelope glycoprotein gp41, CD5, CD23, CD30, HERV-K, IL-1 IRalpha, kappa chain, lambda chain, CSPG4, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD70, TROP-2, CD99, p53, mutated p53, Ras, mutated ras, c-Myc, cytoplasmic serine/threonine kinases (e.g., A-Raf,
  • one of the one or more antigen recognition domains is CD19 FMC63, GD2 14G2a, or a Designed Ankyrin Repeat Protein.
  • the connector domain comprises all or a portion of the hinge domains of CD28, CD8ot, CD3 ⁇ , CD8b, CD4, 41bb, IgG4, IgGl, or IgG2, such as SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.
  • the transmembrane domain comprises all or a portion of the transmembrane domain CD28, CD8a, DAP 10, CD3z, CD8a, CD28, CD8b, 41bb, CD40, CD4, CD3e, CD3g, or CD3d, such as SEQ ID NO: 6.
  • one of one or more costimulatory domains comprise all or part of wildtype or mutant signaling domains from CD28, 41BB or 4-1BB (CD137), ICOS, CD27, CD40, 0X40 (CD134), DAP12, or Myd88, such as SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11.
  • the intracellular signaling domain comprises the activating domains from CD3( ⁇ , DAP10, DAP12, 2B4, CD3g, CD3d, CD3e, CD79a, CD79b, or FcRgamma.
  • one of the one or more unique intracellular signaling domains comprises ZAP70, SLP76, PLCyl, or LAT.
  • one or more of the intracellular signaling domains compromise Src family kinases suck as Lek.
  • one of the one or more intracellular signaling domains comprises SEQ ID NO: 12 or SEQ ID NO: 13.
  • the antigenrecognition domains prior to ligation the antigenrecognition domains, the connector domain, the transmembrane domain, the costimulatory domain, the intracellular signaling domain undergo Type Ils digestion.
  • the ligation can be blunt end ligation.
  • the resulting CAR construct does not comprise any spacers or unnatural amino acid sequences between any of the domains. .
  • Figs. 1 and lb shows an example method of combinatorial serial assembly of barcoded CAR domains.
  • Fig. la. illustrates that Type Ils restriction enzymes can be used to seamlessly fuse protein-coding sequences as they cut a distance away from their recognition sites.
  • the overhang sites can be generated at amino acid residues shared between protein coding segments, as when mutagenizing a single protein or closely related protein family.
  • small linkers such as 1-2 alanine residues can be added (middle).
  • the overhangs can be designed so they can be blunted by T4 polymerase (right), while keeping the coding sequences unchanged.
  • the fragments can be directionally cloned in a blunt/sticky end hybrid ligation reaction.
  • Fig. lb. shows sub-pools were generated for each CAR domain (scFv, connector, transmembrane, costimulatory and activating domain) by cloning each into an entry vector with a barcode sequence, one barcode per individual domain.
  • each was digested with a Type Ils restriction enzyme, blunt-end fixed, digested by either Mfel or EcoRI then ligated in a hybrid sticky-end/blunt reaction. Dual antibiotic selection improves generation of desired ligation product. This allows seamless assembly of chimeric protein domains independently of the Type Ils overhangs.
  • the costimulatory domain sub-pool was assembled twice.
  • Figs. 2a-d show validation of an example assembled barcoded CAR library.
  • Fig. 2c illustrates CAR domain frequencies in the library show up to 2-10-fold differences between domains within a sub-pool, predominantly in the costimulatory domains.
  • Fig. 2d is the cumulative distribution of CD 19 and GD2-CAR construct barcodes reads determined by NGS, ⁇ 13 million reads per library. Fully assembled CARs constitute 95-97% of all reads.
  • Figs. 3a-d show that example GD2 and CD19 expansion library screens reveal no difference between 2 nd and 3 rd generation CARs.
  • Figs. 3a-d are analysis of screened CARs with CD3fwt grouped based on second- or third-generation costimulatory domain architecture as described in Fig. 2. There is no significant difference between these CAR architectures for either CD 19 or GD2-CARs (Mann-Whitney test).
  • Figs. 4a-d illustrate that GD2 and CD 19 library screens showed preference for 4- 1BB costimulatory domain at the membrane proximal position.
  • Fig. 4A is relative expansion of CD19 with a variety of domains
  • Fig. 4b is relative expansion of GD2 with a variety of domains
  • Fig. 4c is CD19 proliferation with a variety of domains
  • Fig. 4D is GD2 with a variety of domains.
  • Fig. 5 illustrates that CD28 CAR has lower surface expression compared to 4-1BB when transduced with high virus concentrations.
  • T cells were transduced with the different CAR constructs at varying virus v/v%, genomic DNA was extracted 10 days later and copy number was determined by qPCR. **** p ⁇ 0.0001; *** p ⁇ 0.001; ** p ⁇ 0.01; Two-way ANOVA.
  • Fig. 9 shows CD19-CARs expansion after repeated antigen stimulation.
  • T cells were transduced with the different CD19-CAR constructs and surviving CAR+ cells were measured via flow cytometry after repeat antigen stimulation, adding Raji cells at a 1 : 1 initial ratio every 2-3 days.
  • Fig. 10 shows in vitro characterization of CD19-CAR constructs exhaustion markers. T cells were transduced with the different CD19-CAR constructs and exposed to 11 cycles of repeat tumor antigen stimulation, then exhaustion markers were measured via flow cytometry.
  • Figs, l la-d shows in vivo characterization of CD19-CAR constructs with second donor.
  • NSG mice were injected intravenously with IxlO 6 luciferase-labeled CD19+ Daudi cells, then three days later received 2xl0 6 CAR+ cells (Fig. I la).
  • Tumor growth was monitored by weekly bioluminescence imaging (Fig. 1 lb).
  • Monitoring tumor bioluminescence demonstrates both IgG4-41BB and IgG4-41BB-CD28 significantly reduce tumor progression, **p ⁇ 0.01, two- way ANOVA with Turkey’s multiple comparison (Fig. 11c).
  • Kaplan-Meier curve was generated from survival of animals in Fig.
  • FIG. 12 is an illustration of an example method of sequential assembly of individual CAR domains using blunt ligation, with each domain having a unique DNA barcode.
  • the schematic shows the seamless combinatorial blunt-end gene assembly protocol developed in which sequentially cloned CAR domains simultaneously generate hundreds of distinct full CAR constructs with unique pre-determined barcodes (scFv, single chain variable fragment; Hin, hinge; TM, transmembrane; Cos, costimulatory domain; Act, activating domain).
  • Fig. 12 also shows example barcodes which are associated with each individual domain.
  • Fig. 13 is a schematic showing the full list of CAR domains tested in an exemplary embodiment, which comprised 180 library members targeting CD 19 and GD2 with varying hinge, costimulatory and activating domains.
  • Fig. 14 shows the tests of the CD19-CAR library, in which gammaretroviral supernatant was produced and MOI determined then transduced into Jurkat cells at a 0.3-0.5 MOI.
  • the transduced cells were co-cultured with Raji cells for six hours, sorted between CD69+ and CD69- cells and CAR barcodes were quantified via NGS. Cell activation was determined for each CAR as the log2 ratio between CD69+ and CD69- normalized reads.
  • Fig. 15 shows that technical replicates of CD 19 CAR library activation show reproducibility of the example CAR screen.
  • Fig. 16 shows the CD 19 CAR library was screened in primary T cells then grouped based on CD3(j domain.
  • the gray shaded area corresponds to two standard deviations from the CD3(jmt mean, shown as a horizontal bar.
  • FIG. 18a is an illustration showing relative expansion of primary T-cells that are transduced with CAR libraries and exposed to irradiated tumors for multiple cycles.
  • Figs. 18b and 18c show primary T cells were transduced with CAR libraries (Fig. 18b, CD 19; Fig. 18c, GD2) then exposed to six (CD 19) or four (GD2) cycles of antigen stimulation with irradiated tumor cells at a 1 :2 effector: tumor ratio every 2-3 days (CD 19: Raji, GD2: CHLA-255) as illustrated in Fig. 18a. Cells were collected on day 0 and at the end of the repeated stimulations, and barcodes were quantified via NGS.
  • Relative expansion was calculated as the log2 normalized ratio of CAR barcode frequencies of total cells between day 0 and the last cycle, and CARs were again grouped based on CD3 ⁇ status. This illustrates that GD2- and CD19-CAR library expansion and proliferation library screens distinguish between CARs containing CD3 wt and CD3 ⁇ mt domains.
  • Fig. 19a is an illustration showing proliferation of T cells that are transduced with CAR libraries and exposed to irradiated tumor for 2 cycles.
  • Fig. 19b is an example graph of CellTrace Violet vs. NGFR.
  • the gray shaded area in Figs. 19c and 19d corresponds to two standard deviations from the CD3( nt mean.
  • Figs. 18b-c and 19c-d illustrate that GD2- and CD19-CAR library expansion and proliferation library screens distinguish between CARs containing CD3 wt and CD3 nt domains.
  • Figs. 20a-j illustrate that hinge domains have different effects on CD 19- and GD2- CARs.
  • Figs. 20a-d are analysis of CD3 wt CARs screened as described in Figs. 18a-c and 19a-d and grouped based on hinge domain. **** p ⁇ 0.0001; *** p ⁇ 0.001; ** p ⁇ 0.01; Kruskal-Wallis test with Dunn's multiple comparisons.
  • Figs. 20e-j are CAR constructs with 4-1BB costimulatory domain, and different scFv and hinge domains were cloned and tested individually in four T cell donors. Figs.
  • Figs. 20i-j show CAR T cells were stimulated with tumor cells every 2-3 days at initial 1 :2 effector: tumor ratio for six cycles and residual tumor was measured by flow cytometry. ****p ⁇ 0.0001, Two- way ANOVA.
  • Figs. 21a-f illustrate that GD2 and CD19 expansion and proliferation library screens show both novel and known CAR architectures.
  • Figs. 21a and 21d are results from relative expansion and proliferation screens shown in XY plot with constructs in purple having p-value ⁇ 0.05;
  • Fig. 21a CD19-CARs, Fig. 21d GD2-CARs.
  • the solid black circles located in the above the dotted line in Figs. 2 lb, c, e, and f in the upper right quadrant in Figs. 21a and d represent CAR structures of interest.
  • FIGs. 22a-d illustrate that GD2-41BB CAR antitumor activity can be optimized by lowering surface expression.
  • Fig. 22b shows GD2-41BB CAR constructs with or without IRES-NGFR were transduced with 100, 10 or 1 %v/v viral supernatant, and CAR MFI/transduction rate was evaluated at 10 days post-transduction.
  • Fig. 22c shows CARs from Fig.
  • Figs. 23a-f illustrate GD24-1BB CAR with screen-optimized expression has potent in vivo antitumor activity.
  • Figs. 23c-f are NSG mice which were injected intravenously with IxlO 6 (Fig. 23c, Fig.
  • Figs. 24a-f show the top hits from CD 19 screen mediate superior in vivo antitumor activity.
  • Fig. 24a shows CAR architectures from CD19 screens which were individually tested, first stained with CellTrace Violet and exposed to two cycles of antigen stimulation with Raji cells.
  • Fig. 24a-f show the top hits from CD 19 screen mediate superior in vivo antitumor activity.
  • Fig. 24a shows CAR architectures from CD19 screens which were individually tested, first stained with CellTrace Violet and exposed to two cycles of antigen stimulation with Raji cells.
  • Fig. 24b shows CD19
  • Figs. 24d-f are NSG mice which were injected intravenously with IxlO 6 luciferase-labeled CD19+ Daudi cells, then three days later received 2xl0 6 CAR+ cells. Tumor growth was monitored by weekly bioluminescence imaging (Fig. 24d).
  • Fig. 24e shows that monitoring tumor bioluminescence demonstrates both IgG4-41BB and IgG4- 41BB-CD28 significantly reduce tumor progression compared to CD8a-41BB, **p ⁇ 0.01, two- way ANOVA with Turkey’s multiple comparison.
  • essentially free in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts.
  • the total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%.
  • Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.
  • the specification “a” or “an” may mean one or more.
  • the words “a” or “an” when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one. Plurality refers to two or more.
  • CAR architecture or “CAR construct” refers to the sequence of different domains which produces a functional CAR; that is, the CAR architecture is a combination of different domains in a certain order.
  • the CAR construct could refer to the DNA sequence or the protein product.
  • CAR architecture comprises, in order, extracellular domains (one or more antigen recognition domains and a hinge or linker domains), a transmembrane domain, and cytoplasmic domains (one or more costimulatory domain, an activating domain and/or other intracellular signaling domains.
  • barcode can refer to a known polynucleotide sequence that allows some feature of a polynucleotide with which the barcode is associated to be identified.
  • the feature of the polynucleotide to be identified is the sample from which the polynucleotide is derived.
  • barcodes are at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more nucleotides in length.
  • barcodes are shorter than 10, 9, 8, 7, 6, 5 or 4 nucleotides in length.
  • barcodes associated with some polynucleotides are of different length than barcodes associated with other polynucleotides.
  • Barcodes can be of sufficient length and comprise sequences that can be sufficiently different to allow the identification of samples based on barcodes with which they are associated. In some cases, each barcode in a plurality of barcodes differ from every other barcode in the plurality at one or more nucleotide positions, such as (in some cases, at least) 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more positions.
  • a barcode can comprise a polynucleotide sequence that serves as an identifier of a specific unique polynucleotide sequence. In embodiments, multiple barcodes can be ligated together and serve as an identifier of multiple individual specific unique polynucleotide sequences.
  • the term “receiving” refers to an acquisition of an element.
  • the acquisition can be direct or indirect. Receipt may occur through a first, second, third or more party.
  • the party may be acting on their or another’s behalf or in concert with another.
  • a first person receiving a DNA construct includes the first person making the DNA construct; a second person making the DNA construct and then giving it to the first person; or, the first person ordering and taking delivery of the DNA construct from a vender. In all three examples, the first person has received the DNA construct. ).
  • the term “uninterrupted” refers to elements that are put together with nothing additional placed in between them.
  • the unique domain barcodes can be ligated together directly, without spacers, making the final combined barcode uninterrupted and combination of CAR domains uniquely identifiable by the final combined barcode.
  • the domains within the CAR construct may also be uninterrupted, meaning CAR construct does not comprise any spacers between any of the domains.
  • a “set,” as used herein refers to any positive whole number of elements including at least two elements.
  • the set is comprised of similar elements, for example, a set of antigen receptor domains could be comprised of a plurality of different polynucleotide sequences for different antigen receptor domains.
  • Embodiments of the disclosure are blunt ligation-based cloning strategy for serial barcoded DNA assembly that eliminates the requirement for compatible junctions.
  • This blunt ligation-based barcoding assembly can be broadly applicable for the synthesis of combinatorial protein libraries seamlessly, without needing to alter their sequences with linkers or spacers.
  • this approach can incorporate any desired antigen-recognition domain as the final cloning step accelerating the evaluation of CAR architectures targeting cancer antigens of interest.
  • certain combinations of specific CAR-T cell domains work together with synergistic results.
  • This blunt ligation-based cloning strategy for serial barcoded DNA assembly eliminates the requirement for compatible junctions, overcoming prior methods which are suboptimal for proteins composed of heterologous domains like CARs, where even small changes such as single alanine insertions can dramatically affect CAR activity. Further, this method allows for interchangeability of all domains, not just antigen recognition domains, signaling domains, and intracellular signaling domains.
  • Methods of the disclosure may be utilized in research, clinical and/or other applications.
  • methods of the disclosure are utilized in screening for effective CARs to be used in CAR T-cell therapy, for example.
  • the party preparing the library may or may not be the party or parties performing the amplification of the library and also may or may not be the party or parties performing analysis of the library, whether amplified or not.
  • a party applying information from the analysis of the amplified library may or may not be the same party that performed the method of preparing the library and/or amplifying part or all of it.
  • Embodiments of the disclosure include a method of screening for CAR-T cell activity comprising the combinatorial cloning of different CAR constructs where each CAR construct is identifiable by a unique polynucleotide barcode.
  • the CAR constructs comprise one antigen recognition (i.e. ScFV) domain, one connector, one transmembrane domain, one or more costimulatory domains and one activating domain, in order.
  • the CAR construct can comprise one or more costimulatory domains.
  • spacers are included between domains. In some embodiments, no spacers are included.
  • each individual domain prior to being ligated together, is comprised on a clone which additionally comprises a barcode specific to the individual domain sequence.
  • the DNA of the domains can then be sequentially ligated together forming a full DNA CAR construct which additionally comprises a unique full construct barcode which comprises each individual barcode of the individual domains, in order. That is, each DNA CAR construct comprises a predetermined DNA barcode corresponding to each of its serially assembled domains sequence.
  • the CAR construct may additionally comprise spacer regions between the domains or may not.
  • Chimeric antigen receptors are engineered transmembrane proteins that combine the specificity of an antigen-specific receptor with a T-cell receptor's function.
  • Fig. 13 illustrates the protein structure of a CAR construct in protein form, including the five described domains.
  • CARs comprise an ectodomain (i.e. costimulatory domain and intracellular signaling domains (i.e. activation) domains), a spacer region, a transmembrane domain and an endodomain (i.e. connector and antigen recognition domains).
  • the ectodomain of a CAR comprises an antigen recognition region which may be an scFV of an antigen-specific antibody.
  • the ectodomain also comprises a signal peptide which directs the nascent protein into the endoplasmic reticulum.
  • the ectodomain comprises a spacer which links the antigen recognition region to the transmembrane domain.
  • any suitable antigen recognition domain may be included in the present method.
  • the antigen targeted by the antigen recognition domain may be associated with certain cancer cells but not associated with non-cancerous cells.
  • Exemplary antigens include, but are not limited to, antigenic molecules from infectious agents, auto-/self-antigens, tumor-Zcancer- associated antigens and tumor neoantigens (Linnemann et al., 2015).
  • An antigen recognition domain to be used in the disclosure need not be discovered yet, but one of skill in the art would understand how to barcode the antigen recognition domain such that it could be used in a barcoded CAR construct, as described herein.
  • the antigen recognition domains listed herein are not an exhaustive list and should be considered examples only.
  • the antigen recognition domain is comprised of two "tandem" CARs, where two antigen-recognition domains are used back-to-back.
  • the antigen recognition domain is comprising of for Bi-CARs, where two different full-length CAR molecules with different antigen-binding domains are tested together.
  • Exemplary antigen recognition domains include those described, for example, in international patent application publication numbers W0200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321, W02013/071154, W02013/123061, U.S. patent application publication numbers US2002131960, US2013287748, US20130149337, U.S.
  • antigen recognition domains include synthetic polypeptides selected for the specific ability to bind to a biological molecule.
  • the antigen recognition domains are CD 19 FMC63 or GD2 14G2a.
  • the antigen recognition domain for use in the barcoded CAR construct is an antibody or functional fragment thereof.
  • the antigen recognition domain is not an antibody or functional fragment thereof but instead is a natural ligand for a receptor.
  • the antigen recognition domain may be a single polypeptide that is bispecific by comprising two or more antigen recognition domains, one of which binds a desired antigen and the other of which binds another, non-identical antigen.
  • the antigen recognition domain may recognize an epitope comprising the shared space between one or more antigens.
  • the binding region can comprise complementary determining regions of a monoclonal antibody, variable regions of a monoclonal antibody and/or antigen binding fragments thereof.
  • that specificity is derived from a peptide (e.g., cytokine) that binds to a receptor.
  • the antigen recognition domain includes an antigen-binding portion or portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb).
  • an antibody molecule such as a single-chain antibody fragment (scFv) derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb).
  • scFv single-chain antibody fragment derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb).
  • the antibody or functional fragment thereof is derived from a known antibody.
  • the antibody may also be one that is generated de novo against the antigen, and the scFv sequence may be obtained, or derived, from such de novo antibodies.
  • the antigens that are recognized by the antigen recognition domain are associated with cancer and include CD19, EBNA, CD123, HER2, CA-125, TRAIL/DR4, CD20, CD70, CD38, CD 123, CLL1, carcinoembryonic antigen, alphafetoprotein, CD56, AKT, Her3, epithelial tumor antigen, CD319 (CS1), ROR1, folate binding protein, HIV-1 envelope glycoprotein gpl20, HIV-1 envelope glycoprotein gp41, CD5, CD23, CD30, HERV-K, IL-l lRalpha, kappa chain, lambda chain, CSPG4, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD70, TROP-2, CD99, p53, mutated p53, Ras, mutated ras, c-Myc, cytoplasmic serine/threonine kinases (
  • sequences for antigens are known in the art, for example, in the GenBank® database: CD19 (Accession No. NG_007275.1), EBNA (Accession No. NG_002392.2), WT1 (Accession No. NG_009272.1), CD123 (Accession No. NC_000023.11), NY-ESO (Accession No. NC_000023.11), EGFRvIII (Accession No. NG_007726.3), MUC1 (Accession No. NG_029383.1), HER2 (Accession No. NG_007503.1), CA-125 (Accession No. NG_055257.1), WT1 (Accession No.
  • Antigen recognition domains may include epitopic regions or epitopic peptides derived from genes mutated in tumor cells or from genes transcribed at different levels in tumor cells compared to normal cells, such as telomerase enzyme, survivin, mesothelin, mutated ras, bcr/abl rearrangement, Her2/neu, mutated or wild-type p53, cytochrome P450 1B1 and abnormally expressed intron sequences, such as N-acetylglucosaminyltransferase-V; clonal rearrangements of immunoglobulin genes generating unique idiotypes in myeloma and B-cell lymphomas; tumor antigens that include epitopic regions or epitopic peptides derived from oncoviral processes, such as human papilloma virus proteins E6 and E7; Epstein bar virus protein LMP2; nonmutated oncofetal proteins with a tumor-selective expression, such as carcinoembryonic
  • Illustrative pathogenic organisms whose antigens to be recognized are contemplated for use in the method described herein include human immunodeficiency virus (HIV), herpes simplex virus (HSV), respiratory syncytial virus (RSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), Influenza A, B and C, vesicular stomatitis virus (VSV), vesicular stomatitis virus (VSV), polyomavirus (e.g., BK virus and JC virus), adenovirus, Staphylococcus species (including Methicillin-resistant Staphylococcus aureus (MRSA)) and Streptococcus species (including Streptococcus pneumoniae).
  • HCV human immunodeficiency virus
  • HSV herpes simplex virus
  • RSV respiratory syncytial virus
  • CMV cytomegalovirus
  • EBV Epstein-Barr virus
  • Influenza A B and C
  • proteins derived from these and other pathogenic microorganisms for use as antigen as described herein, and nucleotide sequences encoding the proteins may be identified in publications and in public databases, such as GENBANK®, SWISS-PROT® and TREMBL®.
  • Exemplary viral antigens also include, but are not limited to, adenovirus polypeptides, alphavirus polypeptides, calicivims polypeptides (e.g., a calicivims capsid antigen), coronavirus polypeptides, distemper virus polypeptides, Ebola virus polypeptides, enterovirus polypeptides, flavivirus polypeptides, hepatitis vims (AE) polypeptides (a hepatitis B core or surface antigen, a hepatitis C vims El or E2 glycoproteins, core or non-structural proteins), herpesvirus polypeptides (including a herpes simplex virus or varicella zoster virus glycoprotein), infectious peritonitis vims polypeptides, leukemia vims polypeptides, Marburg vims polypeptides, orthomyxovirus polypeptides, papilloma vims polypeptides, parain
  • the antigen may be a bacterial antigen.
  • a bacterial antigen of interest may be a secreted polypeptide.
  • bacterial antigens include antigens that have a portion or portions of the polypeptide exposed on the outer cell surface of the bacteria.
  • the antigen may be a fungal antigen.
  • the antigen bay be a protozoan parasite antigen.
  • the connector domain is a hinge domain.
  • a connector domain to be used in the disclosure need not be discovered yet, but given this disclosure, one of skill in the art would understand how to barcode the connector domain as described herein such that it could be used in a barcoded CAR construct.
  • the connector domain comprises all or a portion of the hinge domains of CD28, CD8a, CD3( ⁇ , CD8b, CD4, 41bb, IgG4, IgGl, or IgG2.
  • connector domains includes hinge domains and linker domains.
  • the connector domain is modified by amino acid substitutions to avoid binding to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
  • the transmembrane (TM) domain is the portion of the CAR which traverses the cell membrane.
  • transmembrane domains for use in CAR constructs and would know to make use of such in the present disclosure.
  • a transmembrane domain to be used in the disclosure need not be discovered yet or yet used in CAR constructions, but given this disclosure, one of skill in the art would understand how to barcode a transmembrane domain as described herein such that it could be used in a barcoded CAR construct.
  • the TM domain comprises a hydrophobic alpha helix.
  • the TM domain comprises all or a portion of the TM domain of CD28.
  • the TM domain comprises all or a portion of the TM domain of CD8a.
  • the TM domain comprises all or a portion of the TM domain of DAP 10.
  • the TM domain comprises all or a portion of the TM domain of CD3z, CD8a, CD28, CD8b, 41bb, CD40, CD4, CD3e, CD3g, or CD3d.
  • the CD28, CD8a or DAP 10 transmembrane domain is modified by amino acid substitutions to avoid binding to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
  • the transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Alternatively the transmembrane domain in some embodiments is synthetic.
  • the synthetic transmembrane domain comprises predominantly hydrophobic residues, such as leucine and valine.
  • a triplet of phenylalanine, tryptophan and valine may be found at each end of a synthetic CD8a, CD28 or DAP 10 transmembrane domain.
  • costimulatory domains for use in CAR constructs and would know to make use of such in the present disclosure.
  • a costimulatory domain to be used in the disclosure need not be discovered yet, but given this disclosure, one of skill in the art would understand how to barcode the costimulatory domain as described herein such that it could be used in a barcoded CAR construct.
  • the costimulatory domain comprises wild type or mutant signaling domains from one or more of CD28, 41BB or 4-1BB (CD137), ICOS, CD27, CD40, 0X40 (CD134), DAP12 or Myd88.
  • the costimulatory domain is modified by amino acid substitutions to modulate their signal transduction qualitatively or quantitatively to induce optimal immune effector cell activity.
  • intracellular signaling domains for use in CAR constructs and would know to make use of such in the present disclosure.
  • An intracellular signaling domain to be used in the disclosure need not be discovered yet, but given this disclosure, one of skill in the art would understand how to barcode the intracellular signaling domain as described herein such that it could be used in a barcoded CAR construct.
  • the intracellular signaling is an activation domain.
  • the intracellular signaling domain is modified by amino acid substitutions to modulate their signal transduction qualitatively or quantitatively to induce optimal immune effector cell activity.
  • the intracellular signaling domain is all or part of a domain that is involved in the immune effector cell activation pathway. In specific embodiments the intracellular signaling domain is all or a party of Lek, Fyn, or LAT.
  • the activation domain comprises the zeta chain of CD3, which comprises three copies of the Immunoreceptor Tyrosine-based Activation Motif (ITAM).
  • ITAM generally comprises a Tyr residue separated by two amino acids from a Leu or He.
  • the IT AMs occur in multiples (at least two), and each ITAM is separated from another by 6-8 amino acids.
  • individual barcoded CAR domains are serially ligated together to form a barcoded CAR construct.
  • Each individual barcoded CAR domain for ligation comprises the DNA of at least the individual CAR domain and the barcode identifying the individual CAR domain.
  • the individual barcoded CAR domain can also comprise restriction sites, selection markers or a combination thereof.
  • Fig. 12 illustrates an example of combinatorial serial assembly of barcoded CAR domains.
  • the CAR domains are ligated together using blunt ligation.
  • Type II restriction enzymes are used to create blunt ends (Fig. la).
  • the costimulatory-activation domain construct is then ligated to a transmembrane construct creating a costimulatory-activation- transmembrane domain construct.
  • the costimulatory-activation-transmembrane domain construct is then ligated to a hinge domain, and this construct is then ligated to an antigen receptor domain creating a full DNA CAR construct.
  • Embodiments of the disclosure include CAR constructs comprising 1, 2, 3 or more stimulatory domains.
  • only one type of antigen recognition domain is comprised in the set of antigen recognition domains.
  • only one transmembrane domain is comprised in the set of transmembrane domains.
  • a set of connectors comprises a plurality of types of connectors.
  • a set of costimulatory domains comprises a plurality of types of connector domains.
  • a set of activation domains comprises a plurality of types of activation domains.
  • the plurality is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 60, 70, 80 90, or more types (individual sequences) of domains in a set of domains.
  • each domain can be identified by and comprises a unique barcode (barcodes shown as 2, 4, 6, 8).
  • barcodes shown as 2, 4, 6, 8 When pooled and ligated together, a new set of Cos-Act constructs result which include each combination of the two domain types with each individual domain sequence identifiable by the now combined barcode (2-4; 2-8; 6-4; and, 6-8). Other domains can then be sequentially ligated to the Cos-Act constructs combinatorially, resulting in a library of all or most of the combinations of different domains possible while being able to identify the domains through the use of the combined barcodes.
  • Embodiments of the disclosure comprise a method of producing a barcoded library of CAR constructs, wherein the sequence of domains in each construct in the library is identifiable through the full barcode of each CAR construct.
  • Methods of making the library include the steps of barcoding each domain within a CAR construct and sequentially ligating the domains forming a barcoded CAR construct.
  • a library is formed by receiving a plurality of uniquely barcoded antigen recognition domains; receiving one or more uniquely barcoded connector domains; receiving one or more uniquely barcoded transmembrane domains; receiving a plurality of uniquely barcoded costimulatory domains; receiving a plurality of uniquely barcoded activating domains; ligating together, in sequence, an antigen recognition domain, the connector, the transmembrane domain, the costimulatory domain, the activating domain, such that each resulting CAR construct comprises one scFv domain, one connector, one transmembrane domain, one or more costimulatory domains and one activating domain and wherein each CAR construct comprises a barcode comprising, in order, the unique barcode from each of the scFc domain, the connector, the transmembrane domain, the one or more costimulatory domains and the activating domain.
  • the CAR construct libraries are transfected into immune cells, such as T-cells.
  • the barcoded CAR constructs may be delivered to the immune cells for testing by any suitable vector, including by a viral vector or by a non-viral vector.
  • viral vectors include at least retroviral, lentiviral, adenoviral or adeno-associated viral vectors.
  • non-viral vectors include at least plasmids, transposons, lipids, nanoparticles and so forth.
  • the barcoded CAR construct can be introduced into immune cells as naked DNA or in a suitable vector.
  • Methods of stably transfecting cells by electroporation using naked DNA are known in the art. See, e.g., U.S. Patent No. 6,410,319.
  • naked DNA generally refers to the DNA encoding a chimeric receptor contained in a plasmid expression vector in proper orientation for expression.
  • a viral vector e.g., a retroviral vector, adenoviral vector, adeno- associated viral vector or lentiviral vector
  • Suitable vectors for use in accordance with the method of the present disclosure are non-replicating in the immune cells.
  • a large number of vectors are known that are based on viruses, where the copy number of the virus maintained in the cell is low enough to maintain the viability of the cell, such as, for example, vectors based on HIV, SV40, EBV, HSV or BPV.
  • the sequence of the open reading frame encoding the chimeric receptor can be obtained from a genomic DNA source, a cDNA source or can be synthesized (e.g., via PCR) or combinations thereof. Depending upon the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination thereof as it is found that introns stabilize the mRNA. Also, it may be further advantageous to use endogenous or exogenous non-coding regions to stabilize the mRNA.
  • immune cells comprising a barcoded CAR construct of the present disclosure may be identified in vitro or in vivo by including a marker in the expression vector.
  • markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector.
  • a selection marker is one that confers a property that allows for selection.
  • a positive selection marker is one in which the presence of the marker allows for its selection while a negative selection marker is one in which its presence prevents its selection.
  • An example of a positive selection marker is a drug resistance marker.
  • PBMCs Peripheral blood mononuclear cells
  • buffy coats Gulf Coast Regional Blood Center
  • Ficoll Ficoll
  • OKT3 and anti- CD28 antibodies BD Bioscience
  • T cells can be maintained in RPMI-10 medium supplemented with lOng/ml IL-7 and lOng/ml IL-15, replenished every 48-72 hours.
  • the CAR immune cells are stimulated prior to testing.
  • the CAR immune cells are stimulated through exposure to an antigen expressing cell, such as a tumor cell, which comprises the antigen to which the antigen recognition domain on the CAR protein binds.
  • the CAR immune cells are exposed to the antigen once or more than once.
  • the CAR immune cells could undergo 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 or 50 cycles of exposure to the antigen.
  • all or a part of the barcoded CAR construct DNA is sequenced. In embodiments, only the barcode is sequenced. Sequencing the barcoded CAR construct DNA allows the barcode to be identified, thus identifying the sequences of the domains contained thereon. In embodiments, all or a part of the CAR construct is amplified prior to sequencing.
  • kits comprising DNA, proteins, virus, immune cells, antibodies, reagents, buffers or a combination thereof is also provided herein.
  • the kit comprises a DNA barcoded CAR library.
  • the kit comprises the DNA of a plurality of uniquely barcoded antigen recognition domains, one or more uniquely barcoded connector domains, one or more uniquely barcoded transmembrane domains; a plurality of uniquely barcoded costimulatory domains, and a plurality of uniquely barcoded activating domains.
  • the article of manufacture or kit can further comprise a package insert comprising instructions for using the kit to produce barcoded CAR architectures. Any of the methods described herein may be included in the article of manufacture or kits.
  • the library is composed of 180 distinct CAR constructs each uniquely barcoded encompassing various hinge, costimulatory and activating domains (Fig. lb). To validate the accuracy of the assembly process, we sequenced plasmid clones and observed that approximately 80% of the generated CARs had barcodes corresponding to the expected protein domains (Fig. 2a).
  • the 4-1BB costimulatory domain had a frequency of 2-10% instead of the theoretically expected 20% if the costimulatory domains were evenly distributed (Fig. 2c). Despite these biases, we detected 88-96% of all expected library members (Fig. 2d).
  • CAR library screening for expansion and proliferation discriminates between active and inactive CARs in primary T cells:
  • hinge domain effect on CAR activity depends on the scFv domain: The combinatorial nature of our screen allowed us to assess how different hinge domains affect the relative expansion and proliferation of T cells expressing CD3 wt CARs.
  • constructs with the IgG4 short hinge mediated higher relative expansion and similar proliferation of transduced T cells compared to constructs with the CD8a hinge (Figs. 20a and 20c).
  • the CD8a hinge outperformed others for both phenotypes (Figs. 20b and 20d).
  • CAR library screens identify both established and novel domain combinations, with a preference for the 4-1BB costimulatory domain:
  • CD19-CARs with the IgG4 hinge domain mediated more robust relative expansion of transduced cells, while the same was true for the CD8a hinge in GD2-CARs.
  • CD19-CARs in the relative expansion screen Fig.
  • the top-performing CAR constructs identified in our GD2 screens featured the 4- 1BB, CD28 and 4-1BB-CD28 costimulatory domains, along with the CD8a hinge.
  • the latter two constructs have already demonstrated some activity in clinical trials, showing the translational potential of this approach.
  • We show that low viral concentrations used for the pooled screening allow for both robust expansion and antitumor activity of cells retrovirally transduced with 4-1BB CARs.
  • Our results suggest there is a threshold level of CAR expression for triggering the deleterious LTR-mediated positive feedback loop. Optimization of CAR expression could improve expansion and antitumor activity of clinical cell therapy products, especially those that initially fail manufacturing.
  • the second-generation GD2 4-1BB and third-generation 4-1BB-CD28 CARs mediated similar in vivo antitumor activity. This finding aligns with the overall results from our expansion and proliferation screens, where 4-1BB had a dominant effect in the membrane- proximal position, as previously reported for the ICOS costimulatory domain.
  • the CD28-containing GD2-CAR mediated the most potent antitumor activity in vivo compared to 4- 1BB despite its relative lower performance in the pooled screens. This may be attributed to the higher levels of cytokine secretion induced by CD28, which is underestimated in pooled screening due to bystander effects.
  • the secretion of cytokines such as IFN-gamma is critical for antitumor activity against solid tumors, which could contribute to the superior in vivo performance mediated by the GD2 CD28 CAR.
  • the IgG4 hinge mediated superior in vivo antitumor activity compared to the clinically validated CD8a-41BB CAR architecture, an observation that initially emerged from the screen domain analysis.
  • the IgG4 and CD8a hinge-containing CARs were individually compared in vitro, and no differences were detected between them.
  • pooled screening may be better suited for comparing different CAR architectures in some cases as it may reduce the experimental variability compared with separately manipulating multiple constructs. This could be particularly true in complex, long-term experiments like serial tumor challenges.
  • the hinge domain can also be critical for in vivo activity. For example, the IgG4 hinge with the long CH2-CH3 spacer interacts with myeloid cells, leading to activation-induced T-cell death and poor in vivo persistence.
  • Each domain sub-pool was first digested with either BbsI or Bsal (NEB) and heat-inactivated. Next, sticky ends were blunted with T4 polymerase and dNTPs (NEB), followed by purification using the Monarch PCR & DNA Cleanup Kit (NEB). The product was digested with either Mfel or EcoRI. Then the sub-pool that provides the backbone was processed using Quick CIP (NEB), and the backbone and insert were gel-purified. The insert was ligated into the backbone using T4 Ligase (NEB), then luL was transformed into 10-beta electrocompetent E. coli cells (NEB).
  • Cancer cell line culture CD19+ Raji cells (gift from Dr. Gianpietro Doth) and CHLA-255 cells were stably transduced with a GFP construct and sorted to obtain pure populations. Raji and Daudi cells were maintained in RPMI-10 media (RPMI-1640 media with 10% FBS and 1% GlutaMAXTM, ThermoFisher) and CHLA-255 cells in IMDM-20 (IMDM media with 20% FBS and 1% GlutaMAXTM). Cell lines were routinely STR fingerprinted and tested for mycoplasma contamination.
  • Retrovirus production and transduction conditions CAR libraries and individual CAR constructs were subcloned into empty SFG gammaretroviral vectors or SFG modified to include an IRES-NGFR sequence.
  • 293T cells were co-transfected with SFG-CAR and packaging plasmids RD114 and pEQ-PAM3.
  • Retrovirus supernatant was collected after 24 and 48 hours, filtered with a 0.45uM filter and flash frozen in an ethanol-dry ice bath.
  • nontissue culture 24-well plates (Falcon) were coated with 7pg/mL Retronectin (Takara) overnight at 4oC.
  • the plates were then washed, retrovirus supernatant was added and plates were centrifuged at 4,000g for 1 hour at room temperature. The supernatant was aspirated, and activated T cells were added at 250,000 cells per well, followed by a 10-minute centrifugation at 1,000g. Transduced cells were expanded for 10 days prior to flow cytometry analysis and tumor co-culture assays.
  • Virus supernatant titer was assessed by serial dilution, and transduction efficiency was measured by NGFR staining.
  • CAR-T cells were stained with anti-scFv, PD-1 and LAG3 antibodies.
  • Cell death was assessed by Annexin V-PE (ThermoFisher) and DAPI staining.
  • CAR surface expression was detected using anti -idiotype 1A7 for the GD2-CAR and anti-FMC63 (Custoscan) for the CD19-CAR.
  • Samples were acquired on an IntelliCyt iQue Screener and analyzed using FlowJo vlO. Cell sorting was performed on a Sony SH800 sorter.
  • CD69 CAR-T cell activation assay T cells or Jurkat cells were transduced with retroviral supernatant at low MOI (1 :30 dilution), then 20xl0 6 T cells transduced with the CAR library were co-cultured in a 6-well G-Rex plate (Wilson Wolf) with tumor cells at a 1 :2 ratio (effectortumor). After six (Jurkat) or 24 hours (T), cells were stained with CD69, NGFR and anti- scFv antibodies. CD69+/- cells were sorted on a Sony SH800 sorter.
  • CellTrace Violet proliferation screening At day 10 post-transduction, 20xl0 6 T cells transduced with CAR library were washed with PBS then resuspended at 10 6 cells/mL to achieve a 1 : 1000 dilution in CellTrace Violet. Cell suspensions were incubated in the dark for five minutes, RPMI-10 medium was added and suspensions were transferred to a 6-well G-Rex plate. Irradiated tumor cells were added 1 :2 (effectortumor). Four days after adding tumors, a second cycle of stimulation was performed with 40xl0 6 irradiated tumor cells. Three days later, cells were stained with anti-scFv and NGFR antibodies, and NGFR+ CellTrace Violet-high and -low cells were sorted on a Sony SH800 sorter.
  • Genomic DNA from CAR-T cell donors was isolated using the DNeasy Blood and Tissue kit (QIAGEN). Quantitative PCR was performed with the KAPA SYBR® FAST qPCR Master Mix (Sigma Aldrich) on a C1000 Touch Thermal Cycler with CFX96 Optical Reaction Module (Bio-Rad) using universal adapter primers. Sample amplification curves were monitored, and PCR was repeated while ensuring that cycle number remained in the exponential phase. Second rounds of both qPCR and PCR were performed using luL of initial PCR product and sample-specific multiplex barcoded primers.
  • DESeq2 was used to normalize reads and calculate foldchange and p-values comparing paired samples.
  • CD3 ⁇ wild type versus mutant analysis only CARs with >2,000 normalized reads and the expected scFv domain were analyzed.
  • CD3( ⁇ wild type CARs were analyzed.
  • Graphs and group statistical analyses were performed in GraphPad Prism 9.
  • IFNy secretion Culture supernatants were collected after 24 hours of CAR-T cell co-culture with tumor cells, and IFNy was measured using the Human IFN gamma ELISA MAX Deluxe Set (BioLegend) following the manufacturer’s protocol. Absorbance was read in a Spark 10M Multimode Plate Reader (Tecan) at 450 nm wavelength.
  • CAR copy number analysis Genomic DNA from four CAR-T cell donors was isolated using the DNeasy Blood and Tissue kit (QIAGEN) 10 days after transduction. Primers specific for the anti-GD2 14g2a scFv were tested for efficiency via standard dilution, and specificity was determined using non-transduced control genomic DNA (Table 2). Standard curves were generated for both GD2 and RPL32. Quantitative PCR was performed with KAPA S YBR® FAST qPCR Master Mix (Sigma Aldrich) on a C 1000 T ouch Thermal Cycler with CFX96 Optical Reaction Module (Bio-Rad). Absolute copy numbers in samples were calculated using standard dilution curves from PCR products, and CAR copy number was calculated as GD2 copy number / (RPL32 copy number x2).
  • mice For neuroblastoma, NOD.Cg-Prkdcscid I12rgtml Wjl/SzJ (NSG) mice (8- to 10-week-old females, The Jackson Laboratory) were injected intravenously with l-2x!0 6 CHLA-255 cells engineered to express a FFluc-GFP fusion protein. Two weeks later, mice received a single intravenous injection of 2xl0 6 CAR-T cells. For CD 19+ lymphoma, mice were injected intravenously with IxlO 6 Daudi cells engineered to express a FFluc-GFP fusion protein. Three days later, mice received a single intravenous injection of 2xl0 6 CAR-T cells.
  • Invariant NKT cells with chimeric antigen receptor provide a novel platform for safe and effective cancer immunotherapy.

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Abstract

A method of refining Chimeric Antigen Receptor (CAR) using barcoded protein domain combination screening. The method can be used to make a library of barcoded CAR constructs. Also included is a library of barcoded CAR constructs.

Description

REFINEMENT OF CAR CONSTRUCTS VIA BARCODED SCREENING
This application claims the benefit of U.S. Provisional Application No. 63/580,800, filed on September 6, 2023, the entity of which is incorporated herein by reference.
BACKGROUND
I. Field
[0001] The present disclosure relates generally to the fields of protein engineering development. More particularly, it concerns methods to develop new chimeric antigen receptor (CAR)-based cancer immunotherapies through combinatorial screening and methods of use thereof.
II. Description of Related Art
[0002] Chimeric antigen receptor (CAR)-based cancer immunotherapy has proven successful in treating hematologic malignancies, leading to complete remission for many patients with refractory hematologic malignancies. CAR-based immunotherapies use synthetic receptors that redirect immune effector cells to target cancer cells based on recognition of extracellular antigens. These receptors consist of several essential components: 1) an extracellular antigenrecognition domain, typically a single-chain variable fragment (scFv); 2) a hinge region (or connector) linking the extracellular domain to the transmembrane domain; 3) a single-pass transmembrane domain; 4) one or two cytoplasmic costimulatory domains (second- or third- generation CARs, respectively) that enhance T cell activation by providing “signal two;” and 5) a cytoplasmic domain containing immunoreceptor tyrosine-based activation motifs (ITAMs), usually but not exclusively from CD3(^, that provides “signal one” and initiates the signaling cascade that leads to T cell activation.
[0003] Evaluating CAR domain combinations is impeded by the complex nature of these synthetic constructs and the increasingly large number of immune receptor domains they can be based on. Traditional testing strategies for new CAR architectures are limited by the time and cost of cloning and testing individual constructs. As such, only a fraction of possible domain combinations have been tested to date. Most current methods suffer from low library coverage due to random assembly biases and loss of low-frequency library members after bottlenecking. Further, current methods are suboptimal for proteins composed of heterologous domains like CARs, where even small changes such as single alanine insertions can dramatically affect CAR activity. Therefore, new methods are needed to effectively and quickly test full-length CAR domain combinations.
SUMMARY
[0004] The present disclosure concerns methods and compositions related to constructing libraries of CAR constructs and the methods of their use.
[0005] An exemplary embodiment of the disclosure is a method of producing CAR constructs comprising the steps of: receiving a set of uniquely barcoded antigen-recognition domains comprising one or more unique antigen-recognition domains; receiving a set of uniquely barcoded connector domains comprising a plurality of unique connector domains; receiving a set of uniquely barcoded transmembrane domains comprising one or more unique transmembrane domains; receiving a set of uniquely barcoded costimulatory domains comprising a plurality of unique costimulatory domains; receiving a set of uniquely barcoded intracellular signaling domains comprising one or more unique intracellular signaling domains; and, ligating together the set of uniquely barcoded antigen-recognition domains, the set of uniquely barcoded connector domains, the set of uniquely barcoded transmembrane domains, the set of uniquely barcoded costimulatory domains, the set of uniquely barcoded costimulatory domains, such that each resulting CAR construct comprises, in order, one or more antigen-recognition domains, one connector domain, one transmembrane domain, zero, one or more costimulatory domains, and zero, one or more intracellular signaling domains and wherein each CAR construct comprises a barcode comprising, in order, the unique barcode from each of the one or more antigen-recognition domains, the unique barcode from the connector domain, the unique barcode from the transmembrane domain, the unique barcode from each of the one or more costimulatory domain, and the unique barcode from each of the one or more intracellular signaling domains. Embodiments of the method additionally comprise one or more of the following steps: the step of transfecting the CAR constructs into immune cells; subjecting the immune cells to antigen stimulation; testing the immune cells for proliferation, surface expression, relative expansion, antitumor activity, antivirus activity, antibacterial activity, antifungal activity, removal of cellular pathogenic state or a combination thereof; selecting the immune cells with the high antitumor activity, antivirus activity, or antibacterial activity; identifying the CAR construct based on the barcode; generating treatment immune cells comprising the CAR domains identified in the CAR construct; and, administering the treatment immune cells to a patient in need thereof. In embodiments the immune cells are T cells, NK cells, NKT cells, gamma-delta T-cells, Macrophages, neutrophils, and iPSC-derived effectors, or a combination thereof. In some embodiments, the CARs are delivered to immune cells in vivo via viruses or mRNA vaccines. In some embodiments, one of the one or more antigen recognition domains binds to CD 19, EBNA, CD123, HER2, CA-125, TRAIL/DR4, CD20, CD70, CD38, CD123, CLL1, carcinoembryonic antigen, alphafetoprotein, CD56, AKT, Her3, epithelial tumor antigen, CD319 (CS1), R0R1, folate binding protein, HIV-1 envelope glycoprotein gpl20, HIV-1 envelope glycoprotein gp41, CD5, CD23, CD30, HERV-K, IL-l lRalpha, kappa chain, lambda chain, CSPG4, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD70, TROP-2, CD99, p53, mutated p53, Ras, mutated ras, c-Myc, cytoplasmic serine/threonine kinases (e.g., A-Raf, B-Raf, and C-Raf, cyclin- dependent kinases), MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MART-1, melanoma-associated antigen, BAGE, DAM-6, -10, GAGE-1, -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, MC1R, mda-7, gp75, GplOO, PSA, PSM, Tyrosinase, tyrosinase-related protein, TRP-1, TRP-2, ART -4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, Phosphoinositide 3-kinases (PI3Ks), TRK receptors, PRAME, P15, RU1, RU2, SART-1, SART-3, Wilms' tumor antigen (WT1), AFP, -catenin/m, Caspase-8/m, CDK-4/m, ELF2M, GnT-V, G250, HAGE, HSP70-2M, HST-2, KIAA0205, MUM-1 , MUM-2, MUM-3, Myosin/m, RAGE, SART-2, TRP-2/INT2, 707-AP, Annexin II, CDC27/m, TPI/mbcr-abl, BCR- ABL, interferon regulatory factor 4 (IRF4), ETV6/AML, LDLR/FUT, Pml/RAR, Tumor- associated calcium signal transducer 1 (TACSTD1) TACSTD2, receptor tyrosine kinases (e.g., Epidermal Growth Factor receptor (EGFR) (in particular, EGFRvIII), platelet derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR)), VEGFR2, cytoplasmic tyrosine kinases (e.g., src-family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transducers and activators of transcription STAT3, STATS, and STATE, hypoxia inducible factors (e.g., HIF-1 and HIF-2), Nuclear Factor-Kappa B (NF-B), Notch receptors (e.g., Notchl- 4), NY ESO 1, c-Met, mammalian targets of rapamycin (mTOR), WNT, extracellular signal- regulated kinases (ERKs), and their regulatory subunits, PMSA, PR-3, MDM2, Mesothelin, renal cell carcinoma-5T4, SM22-alpha, carbonic anhydrases I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL/AML1, GD2, proteinase3, hTERT, sarcoma translocation breakpoints, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin Bl, polysialic acid, MYCN, RhoC, GD3, fucosyl GM1, mesothelian, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SAGE, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumain, TIE2, Page , MAD-CT-1, FAP, MAD-CT-2, fos related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNCI, and LRRN1. In additional embodiments, one of the one or more unique antigen recognition domains from antibodies such as CD19 FMC63, GD2 14G2a, or a Designed Ankyrin Repeat Protein. In some embodiments, one of the plurality of unique connector domains comprises all or a portion of the hinge domains of CD28, CD8a, CD3(^, CD8b, CD4, 41bb, IgG4, IgGl, or IgG2. In specific embodiments, one of the plurality of unique connector domains comprises SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO:5. In specific embodiments, one the one or more unique transmembrane domains comprises all or a portion of the transmembrane domain CD28, CD8ot, DAP 10, CD3z, CD8a, CD28, CD8b, 41bb, CD40, CD4, CD3e, CD3g, or CD3d. In some embodiments, one of the one or more unique transmembrane domains comprises SEQ ID NO: 6. In an embodiment, one of the plurality of unique costimulatory domains comprises all or part of wildtype or mutant signaling domains from one or more of CD28, 41BB or 4-1BB (CD137), ICOS, CD27, CD40, 0X40 (CD134), DAP12, or Myd88. In specific embodiments, one of the plurality of unique costimulatory domains comprises SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 . In some embodiments, one or more intracellular signaling domains comprises the activating domains from CD3(^, DAP10, DAP12, 2B4, CD3g, CD3d, CD3e, CD79a, CD79b, or FcRgamma. In embodiments, one of the one or more unique intracellular signaling domains comprises ZAP70, SLP76, PLCyl, or LAT. In some embodiments, one or more of the intracellular signaling domains compromise Src family kinases suck as Lek. In specific embodiments, one of the one or more intracellular signaling domains comprises SEQ ID NO: 12 or SEQ ID NO: 13. In some embodiments, prior to ligation the antigen-recognition domains, the connector domain, the transmembrane domain, the costimulatory domain, the intracellular signaling domain undergo Type Ils digestion. The ligation can be blunt end ligation. In some embodiments, the resulting CAR construct does not comprise any spacers or unnatural amino acid sequences between any of the domains. [0006] Another exemplary embodiment of the disclosure is a library of barcoded CAR constructs comprising: a plurality of CAR constructs comprising one or more antigen-recognition domains, one connector domain, one transmembrane domain, zero, one or more costimulatory domains, and zero, one or more intracellular signaling domain and a unique barcode comprising, in order, an antigen-recognition domain identifying barcode, a connector domain identifying barcode, a transmembrane domain identifying barcode, one or more costimulatory domain identifying barcodes, and an intracellular signaling domain identifying barcode. In embodiments, one of the one or more antigen recognition domain binds to CD 19, EBNA, CD 123, HER2, CA- 125, TRAIL/DR4, CD20, CD70, CD38, CD123, CLL1, carcinoembryonic antigen, alphafetoprotein, CD56, AKT, Her3, epithelial tumor antigen, CD319 (CS1), ROR1, folate binding protein, HIV-1 envelope glycoprotein gpl20, HIV-1 envelope glycoprotein gp41, CD5, CD23, CD30, HERV-K, IL-1 IRalpha, kappa chain, lambda chain, CSPG4, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD70, TROP-2, CD99, p53, mutated p53, Ras, mutated ras, c-Myc, cytoplasmic serine/threonine kinases (e.g., A-Raf, B-Raf, and C-Raf, cyclin-dependent kinases), MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE- A 10, MAGE- A12, MART-1, melanoma-associated antigen, BAGE, DAM-6, -10, GAGE-1, -2, -8, GAGE-3, - 4, -5, -6, -7B, NA88-A, MC1R, mda-7, gp75, GplOO, PSA, PSM, Tyrosinase, tyrosinase-related protein, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, Phosphoinositide 3-kinases (PI3Ks), TRK receptors, PRAME, P15, RU1, RU2, SART-1, SART- 3, Wilms' tumor antigen (WT1), AFP, -catenin/m, Caspase-8/m, CDK-4/m, ELF2M, GnT-V, G250, HAGE, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, Myosin/m, RAGE, SART-2, TRP-2/INT2, 707-AP, Annexin II, CDC27/m, TPI/mbcr-abl, BCR-ABL, interferon regulatory factor 4 (IRF4), ETV6/AML, LDLR/FUT, Pml/RAR, Tumor-associated calcium signal transducer 1 (TACSTD1) TACSTD2, receptor tyrosine kinases (e.g., Epidermal Growth Factor receptor (EGFR) (in particular, EGFRvIII), platelet derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR)), VEGFR2, cytoplasmic tyrosine kinases (e g., src-family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transducers and activators of transcription STAT3, STATS, and STATE, hypoxia inducible factors (e.g., HIF-1 and HIF-2), Nuclear Factor-Kappa B (NF-B), Notch receptors (e.g., Notchl-4), NY ESO 1, c-Met, mammalian targets of rapamycin (mTOR), WNT, extracellular signal-regulated kinases (ERKs), and their regulatory subunits, PMSA, PR-3, MDM2, Mesothelin, renal cell carcinoma- T4, SM22- alpha, carbonic anhydrases I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL/AML1 , GD2, proteinase3, hTERT, sarcoma translocation breakpoints, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin Bl, polysialic acid, MYCN, RhoC, GD3, fucosyl GM1, mesothelian, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SAGE, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD- CT-2, fos related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNCI, and LRRNl. In some embodiments, one of the one or more antigen recognition domains is CD19 FMC63, GD2 14G2a, or a Designed Ankyrin Repeat Protein. In specific embodiments, the connector domain comprises all or a portion of the hinge domains of CD28, CD8ot, CD3^, CD8b, CD4, 41bb, IgG4, IgGl, or IgG2, such as SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In specific embodiments, the transmembrane domain comprises all or a portion of the transmembrane domain CD28, CD8a, DAP 10, CD3z, CD8a, CD28, CD8b, 41bb, CD40, CD4, CD3e, CD3g, or CD3d, such as SEQ ID NO: 6. In embodiments, one of one or more costimulatory domains comprise all or part of wildtype or mutant signaling domains from CD28, 41BB or 4-1BB (CD137), ICOS, CD27, CD40, 0X40 (CD134), DAP12, or Myd88, such as SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11. In some embodiments, the intracellular signaling domain comprises the activating domains from CD3(^, DAP10, DAP12, 2B4, CD3g, CD3d, CD3e, CD79a, CD79b, or FcRgamma. In embodiments, one of the one or more unique intracellular signaling domains comprises ZAP70, SLP76, PLCyl, or LAT. In some embodiments, one or more of the intracellular signaling domains compromise Src family kinases suck as Lek. In specific embodiments, one of the one or more intracellular signaling domains comprises SEQ ID NO: 12 or SEQ ID NO: 13. In some embodiments, prior to ligation the antigenrecognition domains, the connector domain, the transmembrane domain, the costimulatory domain, the intracellular signaling domain undergo Type Ils digestion. The ligation can be blunt end ligation. In some embodiments, the resulting CAR construct does not comprise any spacers or unnatural amino acid sequences between any of the domains. .
[0007] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
[0009] Figs. 1 and lb shows an example method of combinatorial serial assembly of barcoded CAR domains. Fig. la. illustrates that Type Ils restriction enzymes can be used to seamlessly fuse protein-coding sequences as they cut a distance away from their recognition sites. In the simplest case (left), the overhang sites can be generated at amino acid residues shared between protein coding segments, as when mutagenizing a single protein or closely related protein family. In cases where the protein sequences to be fused do not have compatible overhangs, small linkers, such as 1-2 alanine residues can be added (middle). Alternatively, the overhangs can be designed so they can be blunted by T4 polymerase (right), while keeping the coding sequences unchanged. The fragments can be directionally cloned in a blunt/sticky end hybrid ligation reaction. Fig. lb. shows sub-pools were generated for each CAR domain (scFv, connector, transmembrane, costimulatory and activating domain) by cloning each into an entry vector with a barcode sequence, one barcode per individual domain. For the assembly of two sub-pools, for example using the costimulatory and activating domains, each was digested with a Type Ils restriction enzyme, blunt-end fixed, digested by either Mfel or EcoRI then ligated in a hybrid sticky-end/blunt reaction. Dual antibiotic selection improves generation of desired ligation product. This allows seamless assembly of chimeric protein domains independently of the Type Ils overhangs. To make a third generation CAR library, the costimulatory domain sub-pool was assembled twice.
[0010] Figs. 2a-d show validation of an example assembled barcoded CAR library. Figs. 2a and 2b illustrate that barcoding accuracy was determined by comparing the CAR coding sequence to its corresponding barcode via Sanger sequencing of CAR library from plasmid clones (Fig. 2a, n=79), Jurkat cell genomic DNA PCRs (Fig. 2b, n= 43). Fig. 2c illustrates CAR domain frequencies in the library show up to 2-10-fold differences between domains within a sub-pool, predominantly in the costimulatory domains. Fig. 2d is the cumulative distribution of CD 19 and GD2-CAR construct barcodes reads determined by NGS, ~13 million reads per library. Fully assembled CARs constitute 95-97% of all reads.
[0011] Figs. 3a-d show that example GD2 and CD19 expansion library screens reveal no difference between 2nd and 3rd generation CARs. Figs. 3a-d are analysis of screened CARs with CD3fwt grouped based on second- or third-generation costimulatory domain architecture as described in Fig. 2. There is no significant difference between these CAR architectures for either CD 19 or GD2-CARs (Mann-Whitney test).
[0012] Figs. 4a-d illustrate that GD2 and CD 19 library screens showed preference for 4- 1BB costimulatory domain at the membrane proximal position. Figs. 4a-d are an analysis of CD3 wt CARs costimulatory domain, relative to a CD28mt negative control. In the case of third- generation CARs the membrane-proximal domain was included. 4-1BB performed best, being significantly better than the CD28mt at mediating GD2 relative expansion (B, p= 0.0088 Kruskal- Wallis test with Dunn’s multiple comparison test). Fig. 4A is relative expansion of CD19 with a variety of domains, Fig. 4b is relative expansion of GD2 with a variety of domains, Fig. 4c is CD19 proliferation with a variety of domains, and Fig. 4D is GD2 with a variety of domains.
[0013] Fig. 5 illustrates that CD28 CAR has lower surface expression compared to 4-1BB when transduced with high virus concentrations. GD2-41BB and CD28 CAR constructs were transduced with 100, 10 or 1 %v/v viral supernatant and CAR MFI/transduction rate was evaluated at 10 days post-transduction by flow cytometry. N=4 donors.
[0014] Figs. 6a and 6b illustrate the role of transduction markers in GD2-CAR expression and antitumor activity. Comparison of how different transduction markers (NGFR vs CD34) and bicistronic linkers (T2A vs IRES) affect GD2-41BB CAR. Fig. 6a expression and Fig. 6b residual CHLA-255 tumor cells after four cycles of tumor antigen stimulation (relative to the IRES-NGFR used in the CAR libraries). N=4 donors, **p<0.01; *p< 0.05; One-way ANOVA with Dunnett's multiple comparisons test. [0015] Fig. 7 illustrates CAR gene copy number in transduced cells at different virus dilutions. T cells were transduced with the different CAR constructs at varying virus v/v%, genomic DNA was extracted 10 days later and copy number was determined by qPCR. **** p<0.0001; *** p<0.001; ** p<0.01; Two-way ANOVA.
[0016] Figs. 8a and b show CD19-CAR constructs relative expression fraction and cell surface expression vary depending on connector and costimulatory domains. T cells were transduced with the different CD19-CAR constructs, and 10 days later, CAR and NGFR surface expression was measured via flow cytometry. Fig. 8a is fraction of transduced (NGFR+) cells expressing CARs compared to the clinically validated CD8a-41BB structure; **** p<0.0001, Oneway ANOVA with Dunnett’s multiple comparison. Fig. 8b is CAR MFI from the CAR+ fraction compared among CD19-CAR architectures; **** p<0.0001, *** p<0.001; ** p<0.01; One-way ANOVA with Turkey’s multiple comparison test.
[0017] Fig. 9 shows CD19-CARs expansion after repeated antigen stimulation. T cells were transduced with the different CD19-CAR constructs and surviving CAR+ cells were measured via flow cytometry after repeat antigen stimulation, adding Raji cells at a 1 : 1 initial ratio every 2-3 days.
[0018] Fig. 10 shows in vitro characterization of CD19-CAR constructs exhaustion markers. T cells were transduced with the different CD19-CAR constructs and exposed to 11 cycles of repeat tumor antigen stimulation, then exhaustion markers were measured via flow cytometry.
[0019] Figs, l la-d shows in vivo characterization of CD19-CAR constructs with second donor. NSG mice were injected intravenously with IxlO6 luciferase-labeled CD19+ Daudi cells, then three days later received 2xl06 CAR+ cells (Fig. I la). Tumor growth was monitored by weekly bioluminescence imaging (Fig. 1 lb). Monitoring tumor bioluminescence demonstrates both IgG4-41BB and IgG4-41BB-CD28 significantly reduce tumor progression, **p<0.01, two- way ANOVA with Turkey’s multiple comparison (Fig. 11c). Kaplan-Meier curve was generated from survival of animals in Fig. I la, *p= 0.01 (log-rank Mantel-Cox test) (Fig. l id). [0020] Fig. 12 is an illustration of an example method of sequential assembly of individual CAR domains using blunt ligation, with each domain having a unique DNA barcode. The schematic shows the seamless combinatorial blunt-end gene assembly protocol developed in which sequentially cloned CAR domains simultaneously generate hundreds of distinct full CAR constructs with unique pre-determined barcodes (scFv, single chain variable fragment; Hin, hinge; TM, transmembrane; Cos, costimulatory domain; Act, activating domain). Fig. 12 also shows example barcodes which are associated with each individual domain.
[0021] Fig. 13 is a schematic showing the full list of CAR domains tested in an exemplary embodiment, which comprised 180 library members targeting CD 19 and GD2 with varying hinge, costimulatory and activating domains.
[0022] Fig. 14 shows the tests of the CD19-CAR library, in which gammaretroviral supernatant was produced and MOI determined then transduced into Jurkat cells at a 0.3-0.5 MOI. The transduced cells were co-cultured with Raji cells for six hours, sorted between CD69+ and CD69- cells and CAR barcodes were quantified via NGS. Cell activation was determined for each CAR as the log2 ratio between CD69+ and CD69- normalized reads.
[0023] Fig. 15 shows that technical replicates of CD 19 CAR library activation show reproducibility of the example CAR screen.
[0024] Fig. 16 shows the CD 19 CAR library was screened in primary T cells then grouped based on CD3(j domain. The gray shaded area corresponds to two standard deviations from the CD3(jmt mean, shown as a horizontal bar.
[0025] Fig. 17 illustrates that CARs with CD3(jwt domain have higher CD69 induction relative to CD3(jmt (p= 0.0003, Mann-Whitney test).
[0026] Fig. 18a is an illustration showing relative expansion of primary T-cells that are transduced with CAR libraries and exposed to irradiated tumors for multiple cycles. Figs. 18b and 18c show primary T cells were transduced with CAR libraries (Fig. 18b, CD 19; Fig. 18c, GD2) then exposed to six (CD 19) or four (GD2) cycles of antigen stimulation with irradiated tumor cells at a 1 :2 effector: tumor ratio every 2-3 days (CD 19: Raji, GD2: CHLA-255) as illustrated in Fig. 18a. Cells were collected on day 0 and at the end of the repeated stimulations, and barcodes were quantified via NGS. Relative expansion was calculated as the log2 normalized ratio of CAR barcode frequencies of total cells between day 0 and the last cycle, and CARs were again grouped based on CD3^ status. This illustrates that GD2- and CD19-CAR library expansion and proliferation library screens distinguish between CARs containing CD3 wt and CD3^mt domains.
[0027] Fig. 19a is an illustration showing proliferation of T cells that are transduced with CAR libraries and exposed to irradiated tumor for 2 cycles. Fig. 19b is an example graph of CellTrace Violet vs. NGFR. T cells were transduced with CD 19- and GD2-CAR libraries (Fig. 19c, CD 19; Fig. 19d, GD2), stained with CellTrace Violet and exposed to two cycles of antigen stimulation. After stimulation, CellTrace-high and -low cells were sorted and sequenced, n= 4 donors, **** p<0.0001, Mann-Whitney test. The gray shaded area in Figs. 19c and 19d corresponds to two standard deviations from the CD3( nt mean. Figs. 18b-c and 19c-d illustrate that GD2- and CD19-CAR library expansion and proliferation library screens distinguish between CARs containing CD3 wt and CD3 nt domains.
[0028] Figs. 20a-j illustrate that hinge domains have different effects on CD 19- and GD2- CARs. Figs. 20a-d are analysis of CD3 wt CARs screened as described in Figs. 18a-c and 19a-d and grouped based on hinge domain. **** p<0.0001; *** p<0.001; ** p<0.01; Kruskal-Wallis test with Dunn's multiple comparisons. Figs. 20e-j are CAR constructs with 4-1BB costimulatory domain, and different scFv and hinge domains were cloned and tested individually in four T cell donors. Figs. 20e-f are CAR MFI comparisons between CARs with indicated hinge domains measured 10 days post-transduction, **p= 0.0080, *p= 0.0390, paired T test. Figs. 20g-h show IFNy secretion ELISA from individual 4-1BB CARs with different hinge domains after 24 hours of co-culture with tumor cells at a 1:2 effector: tumor ratio *p= 0.0260, paired T test. Figs. 20i-j show CAR T cells were stimulated with tumor cells every 2-3 days at initial 1 :2 effector: tumor ratio for six cycles and residual tumor was measured by flow cytometry. ****p <0.0001, Two- way ANOVA.
[0029] Figs. 21a-f illustrate that GD2 and CD19 expansion and proliferation library screens show both novel and known CAR architectures. Figs. 21a and 21d are results from relative expansion and proliferation screens shown in XY plot with constructs in purple having p-value <0.05; Fig. 21a CD19-CARs, Fig. 21d GD2-CARs. Volcano plots for CAR library screen results for CD19-CAR relative expansion Fig. 21b and proliferation Fig. 21 c, and GD2-CAR relative expansion Fig. 21e and proliferation Fig. 21f, horizontal dotted lines indicate p= 0.05. The solid black circles located in the above the dotted line in Figs. 2 lb, c, e, and f in the upper right quadrant in Figs. 21a and d represent CAR structures of interest.
[0030] Figs. 22a-d illustrate that GD2-41BB CAR antitumor activity can be optimized by lowering surface expression. Fig. 22a shows CAR architectures from GD2 screens were individually tested, and residual CHLA-255 tumor cells were measured after four cycles of repeated antigen stimulation. **** p<0.0001, *** p=0.0001 One-way ANOVA, n = 4. Data is represented as individual values and mean (horizontal line). Fig. 22b shows GD2-41BB CAR constructs with or without IRES-NGFR were transduced with 100, 10 or 1 %v/v viral supernatant, and CAR MFI/transduction rate was evaluated at 10 days post-transduction. Fig. 22c shows CARs from Fig. 22b which were exposed to five cycles of repeated antigen stimulation with CHLA-255 cells at an initial 1 :2 effector: tumor ratio. Persisting CAR+ cells and residual tumor cells were simultaneously measured by flow cytometry. Fig. 22d shows PD-1/LAG3 immunofluorescence staining of T cells expressing 4-1BB CAR with or without IRES-NGFR transduced with 100, 10 or 1% virus v/v after three cycles of repeated antigen stimulation. Data is represented as mean ± SD. **** p<0.0001; *** p<0.001; Two-way ANOVA; n = 4.
[0031] Figs. 23a-f illustrate GD24-1BB CAR with screen-optimized expression has potent in vivo antitumor activity. T cells transduced with 100, 10 or 1 %v/v viral supernatant for constructs with either GD24-1BB or CD28 costimulation; after 10 days scFv+ cells (Fig. 23a) and Annexin V+/DAPI+ cells (Fig. 23b) were counted. *** p<0.001, ** p<0.01, *p < 0.05, Two-way ANOVA. Data is represented as mean ± SD. Figs. 23c-f are NSG mice which were injected intravenously with IxlO6 (Fig. 23c, Fig. 23d) or 2xl06 (Fig. 23e, Fig. 231) luciferase-labeled CHLA-255 cells, then two weeks later received 2xl06 CAR+ cells with CD28 transduced with 100% v/v viral supernatant and 4-1BB 10% v/v. Tumor growth was monitored by weekly bioluminescence imaging. Kaplan-Meier curves were generated from survival of animals in Fig. 23c and Fig. 23e. *p= 0.0225 (log-rank Mantel-Cox test).
[0032] Figs. 24a-f show the top hits from CD 19 screen mediate superior in vivo antitumor activity. Fig. 24a shows CAR architectures from CD19 screens which were individually tested, first stained with CellTrace Violet and exposed to two cycles of antigen stimulation with Raji cells. Fig. 24b shows CD19-CARs which were exposed to 11 cycles of antigen stimulation with Raji tumor cells at a 1 : 1 effector: tumor ratio every 2-3 days, then the area under the curve of the remaining CAR+ cells at the end of each cycle was calculated (One-way ANOVA with Turkey’s multiple comparison, *** p<0.001, ** p<0.01; n=4 donors). Fig. 24c shows residual Raji cells after 11 cycles of tumor exposure (N=4 donors). Figs. 24d-f are NSG mice which were injected intravenously with IxlO6 luciferase-labeled CD19+ Daudi cells, then three days later received 2xl06 CAR+ cells. Tumor growth was monitored by weekly bioluminescence imaging (Fig. 24d). Fig. 24e shows that monitoring tumor bioluminescence demonstrates both IgG4-41BB and IgG4- 41BB-CD28 significantly reduce tumor progression compared to CD8a-41BB, **p<0.01, two- way ANOVA with Turkey’s multiple comparison. Fig. 24f is a Kaplan-Meier curve generated from survival of animals in Fig. 24d which show IgG4-41BB lead to improved survival compared with CD8a-41BB, *p= 0.02 (log-rank Mantel-Cox test).
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
I. Definitions
[0033] As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.
[0034] As used herein, the specification “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one. Plurality refers to two or more.
[0035] The use of the term “or” in the claim(s) is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” As used herein, “another” may mean at least a second or more. The terms “about”, “substantially” and “approximately” mean, in general, the stated value plus or minus 5%. [0036] As used herein, “CAR architecture” or “CAR construct” refers to the sequence of different domains which produces a functional CAR; that is, the CAR architecture is a combination of different domains in a certain order. The CAR construct could refer to the DNA sequence or the protein product. The different domains may be directly connected to each other or may be spaced apart. In embodiments, CAR architecture comprises, in order, extracellular domains (one or more antigen recognition domains and a hinge or linker domains), a transmembrane domain, and cytoplasmic domains (one or more costimulatory domain, an activating domain and/or other intracellular signaling domains.
[0037] The term “barcode” can refer to a known polynucleotide sequence that allows some feature of a polynucleotide with which the barcode is associated to be identified. In some cases, the feature of the polynucleotide to be identified is the sample from which the polynucleotide is derived. In some cases, barcodes are at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more nucleotides in length. In some cases, barcodes are shorter than 10, 9, 8, 7, 6, 5 or 4 nucleotides in length. In some cases, barcodes associated with some polynucleotides are of different length than barcodes associated with other polynucleotides. Barcodes can be of sufficient length and comprise sequences that can be sufficiently different to allow the identification of samples based on barcodes with which they are associated. In some cases, each barcode in a plurality of barcodes differ from every other barcode in the plurality at one or more nucleotide positions, such as (in some cases, at least) 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more positions. A barcode can comprise a polynucleotide sequence that serves as an identifier of a specific unique polynucleotide sequence. In embodiments, multiple barcodes can be ligated together and serve as an identifier of multiple individual specific unique polynucleotide sequences.
[0038] As used herein, the term “receiving” refers to an acquisition of an element. The acquisition can be direct or indirect. Receipt may occur through a first, second, third or more party. The party may be acting on their or another’s behalf or in concert with another. For example, a first person receiving a DNA construct includes the first person making the DNA construct; a second person making the DNA construct and then giving it to the first person; or, the first person ordering and taking delivery of the DNA construct from a vender. In all three examples, the first person has received the DNA construct. ). [0039] As used herein, the term “uninterrupted” refers to elements that are put together with nothing additional placed in between them. For example, when ligating the DNA of CAR domains together where each individual domain has a unique DNA barcode, the unique domain barcodes can be ligated together directly, without spacers, making the final combined barcode uninterrupted and combination of CAR domains uniquely identifiable by the final combined barcode. As another example, the domains within the CAR construct may also be uninterrupted, meaning CAR construct does not comprise any spacers between any of the domains.
[0040] A “set,” as used herein refers to any positive whole number of elements including at least two elements. In embodiments, the set is comprised of similar elements, for example, a set of antigen receptor domains could be comprised of a plurality of different polynucleotide sequences for different antigen receptor domains.
II. CAR T-Cell Screening
[0041] Embodiments of the disclosure are blunt ligation-based cloning strategy for serial barcoded DNA assembly that eliminates the requirement for compatible junctions. This blunt ligation-based barcoding assembly can be broadly applicable for the synthesis of combinatorial protein libraries seamlessly, without needing to alter their sequences with linkers or spacers. In particular embodiments, this approach can incorporate any desired antigen-recognition domain as the final cloning step accelerating the evaluation of CAR architectures targeting cancer antigens of interest. In embodiments of the disclosure, certain combinations of specific CAR-T cell domains work together with synergistic results. This blunt ligation-based cloning strategy for serial barcoded DNA assembly eliminates the requirement for compatible junctions, overcoming prior methods which are suboptimal for proteins composed of heterologous domains like CARs, where even small changes such as single alanine insertions can dramatically affect CAR activity. Further, this method allows for interchangeability of all domains, not just antigen recognition domains, signaling domains, and intracellular signaling domains.
[0042] Methods of the disclosure may be utilized in research, clinical and/or other applications. In particular embodiments, methods of the disclosure are utilized in screening for effective CARs to be used in CAR T-cell therapy, for example. In some cases, the party preparing the library may or may not be the party or parties performing the amplification of the library and also may or may not be the party or parties performing analysis of the library, whether amplified or not. A party applying information from the analysis of the amplified library may or may not be the same party that performed the method of preparing the library and/or amplifying part or all of it.
A. CAR Construct Domains
[0043] Embodiments of the disclosure include a method of screening for CAR-T cell activity comprising the combinatorial cloning of different CAR constructs where each CAR construct is identifiable by a unique polynucleotide barcode. In embodiments, the CAR constructs comprise one antigen recognition (i.e. ScFV) domain, one connector, one transmembrane domain, one or more costimulatory domains and one activating domain, in order. In embodiments, the CAR construct can comprise one or more costimulatory domains. In embodiments, spacers are included between domains. In some embodiments, no spacers are included. In embodiments, prior to being ligated together, each individual domain is comprised on a clone which additionally comprises a barcode specific to the individual domain sequence. The DNA of the domains can then be sequentially ligated together forming a full DNA CAR construct which additionally comprises a unique full construct barcode which comprises each individual barcode of the individual domains, in order. That is, each DNA CAR construct comprises a predetermined DNA barcode corresponding to each of its serially assembled domains sequence. The CAR construct may additionally comprise spacer regions between the domains or may not.
[0044] Chimeric antigen receptors (CARs) are engineered transmembrane proteins that combine the specificity of an antigen-specific receptor with a T-cell receptor's function. Fig. 13 illustrates the protein structure of a CAR construct in protein form, including the five described domains. In general, CARs comprise an ectodomain (i.e. costimulatory domain and intracellular signaling domains (i.e. activation) domains), a spacer region, a transmembrane domain and an endodomain (i.e. connector and antigen recognition domains). In exemplary aspects, the ectodomain of a CAR comprises an antigen recognition region which may be an scFV of an antigen-specific antibody. In some embodiments, the ectodomain also comprises a signal peptide which directs the nascent protein into the endoplasmic reticulum. In exemplary aspects, the ectodomain comprises a spacer which links the antigen recognition region to the transmembrane domain. Certain embodiments of the present disclosure concern the use of nucleic acids, including nucleic acids encoding a barcoded CAR polypeptide construct.
1. Antigen recognition domains
[0045] Any suitable antigen recognition domain may be included in the present method. In some cases, the antigen targeted by the antigen recognition domain may be associated with certain cancer cells but not associated with non-cancerous cells. Exemplary antigens include, but are not limited to, antigenic molecules from infectious agents, auto-/self-antigens, tumor-Zcancer- associated antigens and tumor neoantigens (Linnemann et al., 2015). One of skill in the art will recognize antigen recognition domains and would know to make use of such in the present disclosure. An antigen recognition domain to be used in the disclosure need not be discovered yet, but one of skill in the art would understand how to barcode the antigen recognition domain such that it could be used in a barcoded CAR construct, as described herein. The antigen recognition domains listed herein are not an exhaustive list and should be considered examples only.
[0046] In embodiments, the antigen recognition domain is comprised of two "tandem" CARs, where two antigen-recognition domains are used back-to-back. In some embodiments, the antigen recognition domain is comprising of for Bi-CARs, where two different full-length CAR molecules with different antigen-binding domains are tested together.
[0047] Exemplary antigen recognition domains, as well as methods for engineering and introducing the receptors in CARs into cells, include those described, for example, in international patent application publication numbers W0200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321, W02013/071154, W02013/123061, U.S. patent application publication numbers US2002131960, US2013287748, US20130149337, U.S. Patent Nos.: 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446, 179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and European patent application number EP2537416, and/or those described by Sadelain et al., 2013; Davila et al., 2013; Turtle et al., 2012; Wu et al., 2012. In embodiments, antigen recognition domains include synthetic polypeptides selected for the specific ability to bind to a biological molecule. In embodiments, the antigen recognition domains are CD 19 FMC63 or GD2 14G2a. [0048] In some embodiments, the antigen recognition domain for use in the barcoded CAR construct is an antibody or functional fragment thereof. In other cases, the antigen recognition domain is not an antibody or functional fragment thereof but instead is a natural ligand for a receptor. The antigen recognition domain may be a single polypeptide that is bispecific by comprising two or more antigen recognition domains, one of which binds a desired antigen and the other of which binds another, non-identical antigen.
[0049] In certain embodiments, the antigen recognition domain may recognize an epitope comprising the shared space between one or more antigens. In certain embodiments, the binding region can comprise complementary determining regions of a monoclonal antibody, variable regions of a monoclonal antibody and/or antigen binding fragments thereof. In another embodiment, that specificity is derived from a peptide (e.g., cytokine) that binds to a receptor.
[0050] It is contemplated that human CAR nucleic acids may be human genes used to enhance cellular immunotherapy for human patients. In a specific embodiment, the disclosure includes a barcoded CAR construct comprising full-length antigen-specific CAR cDNA or coding region. The antigen binding regions or domain can comprise a fragment of the VH and Vi, chains of a single-chain variable fragment (scFv) derived from a particular human monoclonal antibody, such as those described in U.S. Patent 7,109,304 incorporated herein by reference. The fragment can also be any number of different antigen recognition domains of a human antigen-specific antibody. In a more specific embodiment, the fragment is an antigen-specific scFv encoded by a sequence that is optimized for human codon usage for expression in human cells.
[0051] In some embodiments, an antigen recognition domain is constructed with specificity for a specific antigen, such as the antigen being expressed on a diseased cell type (a cancer cell or cell infected with an infectious agent). One of skill in the art is able to generate antibodies, including scFvs against the antigen based on knowledge at least of the polypeptide and routine practices although numerous anti-antigen scFvs and monoclonal antibodies may already be present in the art. In some embodiments, the antigen-specific scFv is an scFV from one or more of antibody clones.
[0052] In some embodiments, the antigen recognition domain includes an antigen-binding portion or portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb). In specific embodiments, the antibody or functional fragment thereof is derived from a known antibody. The antibody may also be one that is generated de novo against the antigen, and the scFv sequence may be obtained, or derived, from such de novo antibodies.
[0053] In particular aspects, the antigens that are recognized by the antigen recognition domain are associated with cancer and include CD19, EBNA, CD123, HER2, CA-125, TRAIL/DR4, CD20, CD70, CD38, CD 123, CLL1, carcinoembryonic antigen, alphafetoprotein, CD56, AKT, Her3, epithelial tumor antigen, CD319 (CS1), ROR1, folate binding protein, HIV-1 envelope glycoprotein gpl20, HIV-1 envelope glycoprotein gp41, CD5, CD23, CD30, HERV-K, IL-l lRalpha, kappa chain, lambda chain, CSPG4, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD70, TROP-2, CD99, p53, mutated p53, Ras, mutated ras, c-Myc, cytoplasmic serine/threonine kinases (e.g., A-Raf, B-Raf, and C-Raf, cyclin-dependent kinases), MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MART-1, melanoma-associated antigen, BAGE, DAM-6, -10, GAGE-1, -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, MC1R, mda-7, gp75, GplOO, PSA, PSM, Tyrosinase, tyrosinase-related protein, TRP- 1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, Phosphoinositide 3 -kinases (PI3Ks), TRK receptors, PRAME, Pl 5, RU1, RU2, SART-1, S ART-3, Wilms' tumor antigen (WT1), AFP, -catenin/m, Caspase-8/m, CDK-4/m, ELF2M, GnT-V, G250, HAGE, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, Myosin/m, RAGE, SART-2, TRP- 2/INT2, 707-AP, Annexin II, CDC27/m, TPI/mbcr-abl, BCR-ABL, interferon regulatory factor 4 (IRF4), ETV6/AML, LDLR/FUT, Pml/RAR, Tumor-associated calcium signal transducer 1 (TACSTD1) TACSTD2, receptor tyrosine kinases (e.g., Epidermal Growth Factor receptor (EGFR) (in particular, EGFRvIII), platelet derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR)), VEGFR2, cytoplasmic tyrosine kinases (e.g., src- family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transducers and activators of transcription STAT3, STATS and STATE, hypoxia inducible factors (e.g., HIF-1 and HIF-2), Nuclear Factor-Kappa B (NF-B), Notch receptors (e. ., Notch 1-4), NY ESO 1, c-Met, mammalian targets of rapamycin (mTOR), WNT, extracellular signal-regulated kinases (ERKs) and their regulatory subunits, PMSA, PR-3, MDM2, Mesothelin, renal cell carcinoma-5 T4, SM22-alpha, carbonic anhydrases I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL/AML1, GD2, proteinase3, hTERT, sarcoma translocation breakpoints, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin Bl, polysialic acid, MYCN, RhoC, GD3, fucosyl GM1, mesothelian, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SAGE, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD- CT-2, fos related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNCI and LRRN1. Examples of sequences for antigens are known in the art, for example, in the GenBank® database: CD19 (Accession No. NG_007275.1), EBNA (Accession No. NG_002392.2), WT1 (Accession No. NG_009272.1), CD123 (Accession No. NC_000023.11), NY-ESO (Accession No. NC_000023.11), EGFRvIII (Accession No. NG_007726.3), MUC1 (Accession No. NG_029383.1), HER2 (Accession No. NG_007503.1), CA-125 (Accession No. NG_055257.1), WT1 (Accession No. NG_009272.1), Mage-A3 (Accession No. NG_013244.1), Mage-A4 (Accession No. NG_013245.1), Mage-AlO (Accession No. NC_000023.11), TRAIL/DR4 (Accession No. NC_000003.12) and/or CEA (Accession No. NC_000019.10).
[0054] Tumor-associated antigens may be derived from prostate, breast, colorectal, lung, pancreatic, renal, mesothelioma, ovarian, liver, brain, bone, stomach, spleen, testicular, cervical, anal, gall bladder, thyroid or melanoma cancers, as examples. Exemplary tumor-associated antigens or tumor cell-derived antigens include MAGE 1, 3 and MAGE 4 (or other MAGE antigens, such as those disclosed in International Patent Publication No. WO 99/40188); PRAME; BAGE; RAGE, Lage (also known as NY ESO 1); SAGE; and HAGE or GAGE. These nonlimiting examples of tumor antigens are expressed in a wide range of tumor types, such as melanoma, lung carcinoma, sarcoma and bladder carcinoma. See, e.g., U.S. Patent No. 6,544,518. Prostate cancer tumor-associated antigens include, for example, prostate specific membrane antigen (PSMA), prostate-specific antigen (PSA), prostatic acid phosphates, NKX3.1 and six- transmembrane epithelial antigen of the prostate (STEAP).
[0055] Other tumor associated antigens include Plu-1, HASH-1, HasH-2, Cripto and Criptin. Additionally, a tumor antigen may be a self-peptide hormone, such as whole length gonadotrophin hormone releasing hormone (GnRH), a short 10 amino acid long peptide, useful in the treatment of many cancers. [0056] Antigen recognition domains may include epitopic regions or epitopic peptides derived from genes mutated in tumor cells or from genes transcribed at different levels in tumor cells compared to normal cells, such as telomerase enzyme, survivin, mesothelin, mutated ras, bcr/abl rearrangement, Her2/neu, mutated or wild-type p53, cytochrome P450 1B1 and abnormally expressed intron sequences, such as N-acetylglucosaminyltransferase-V; clonal rearrangements of immunoglobulin genes generating unique idiotypes in myeloma and B-cell lymphomas; tumor antigens that include epitopic regions or epitopic peptides derived from oncoviral processes, such as human papilloma virus proteins E6 and E7; Epstein bar virus protein LMP2; nonmutated oncofetal proteins with a tumor-selective expression, such as carcinoembryonic antigen and alphafetoprotein.
[0057] In other embodiments, an antigen is obtained or derived from an infectious agent, including a pathogenic microorganism or from an opportunistic pathogenic microorganism (also called herein an infectious disease microorganism), such as a virus, fungus, parasite, protozoan and bacterium. In certain embodiments, antigens derived from such a microorganism include full- length proteins. Antigen recognition domains can be engineered to recognize the antigens from these infectious agents.
[0058] Illustrative pathogenic organisms whose antigens to be recognized are contemplated for use in the method described herein include human immunodeficiency virus (HIV), herpes simplex virus (HSV), respiratory syncytial virus (RSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), Influenza A, B and C, vesicular stomatitis virus (VSV), vesicular stomatitis virus (VSV), polyomavirus (e.g., BK virus and JC virus), adenovirus, Staphylococcus species (including Methicillin-resistant Staphylococcus aureus (MRSA)) and Streptococcus species (including Streptococcus pneumoniae). As would be understood by the skilled person, proteins derived from these and other pathogenic microorganisms for use as antigen as described herein, and nucleotide sequences encoding the proteins, may be identified in publications and in public databases, such as GENBANK®, SWISS-PROT® and TREMBL®.
[0059] Exemplary viral antigens also include, but are not limited to, adenovirus polypeptides, alphavirus polypeptides, calicivims polypeptides (e.g., a calicivims capsid antigen), coronavirus polypeptides, distemper virus polypeptides, Ebola virus polypeptides, enterovirus polypeptides, flavivirus polypeptides, hepatitis vims (AE) polypeptides (a hepatitis B core or surface antigen, a hepatitis C vims El or E2 glycoproteins, core or non-structural proteins), herpesvirus polypeptides (including a herpes simplex virus or varicella zoster virus glycoprotein), infectious peritonitis vims polypeptides, leukemia vims polypeptides, Marburg vims polypeptides, orthomyxovirus polypeptides, papilloma vims polypeptides, parainfluenza vims polypeptides (e. , the hemagglutinin and neuraminidase polypeptides), paramyxovirus polypeptides, parvovirus polypeptides, pestivims polypeptides, picorna vims polypeptides (e.g., a poliovims capsid polypeptide), pox vims polypeptides (e.g., a vaccinia vims polypeptide), rabies vims polypeptides e.g., a rabies vims glycoprotein G), reovims polypeptides, retrovirus polypeptides and rotavirus polypeptides.
[0060] In certain embodiments, the antigen may be a bacterial antigen. In certain embodiments, a bacterial antigen of interest may be a secreted polypeptide. In other certain embodiments, bacterial antigens include antigens that have a portion or portions of the polypeptide exposed on the outer cell surface of the bacteria. In embodiments, the antigen may be a fungal antigen. In embodiments, the antigen bay be a protozoan parasite antigen.
2. Connector Domains
[0061] One of skill in the art will recognize connector domains for use in CAR constructs and would know to make use of such in the present disclosure. In specific embodiment, the connector domain is a hinge domain. A connector domain to be used in the disclosure need not be discovered yet, but given this disclosure, one of skill in the art would understand how to barcode the connector domain as described herein such that it could be used in a barcoded CAR construct. In exemplary aspects, the connector domain comprises all or a portion of the hinge domains of CD28, CD8a, CD3(^, CD8b, CD4, 41bb, IgG4, IgGl, or IgG2. As used herein, connector domains includes hinge domains and linker domains. In some instances, the connector domain is modified by amino acid substitutions to avoid binding to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. 3. Transmembrane Domain
[0062] The transmembrane (TM) domain is the portion of the CAR which traverses the cell membrane. One of skill in the art will recognize transmembrane domains for use in CAR constructs and would know to make use of such in the present disclosure. A transmembrane domain to be used in the disclosure need not be discovered yet or yet used in CAR constructions, but given this disclosure, one of skill in the art would understand how to barcode a transmembrane domain as described herein such that it could be used in a barcoded CAR construct.
[0063] In exemplary aspects, the TM domain comprises a hydrophobic alpha helix. In exemplary aspects, the TM domain comprises all or a portion of the TM domain of CD28. In exemplary aspects, the TM domain comprises all or a portion of the TM domain of CD8a. In embodiments, the TM domain comprises all or a portion of the TM domain of DAP 10. In embodiments, the TM domain comprises all or a portion of the TM domain of CD3z, CD8a, CD28, CD8b, 41bb, CD40, CD4, CD3e, CD3g, or CD3d.
[0064] In some instances, the CD28, CD8a or DAP 10 transmembrane domain is modified by amino acid substitutions to avoid binding to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. The transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Alternatively the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues, such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine may be found at each end of a synthetic CD8a, CD28 or DAP 10 transmembrane domain.
4. Costimulatory Domains
[0065] One of skill in the art will recognize costimulatory domains for use in CAR constructs and would know to make use of such in the present disclosure. A costimulatory domain to be used in the disclosure need not be discovered yet, but given this disclosure, one of skill in the art would understand how to barcode the costimulatory domain as described herein such that it could be used in a barcoded CAR construct. In exemplary aspects, the costimulatory domain comprises wild type or mutant signaling domains from one or more of CD28, 41BB or 4-1BB (CD137), ICOS, CD27, CD40, 0X40 (CD134), DAP12 or Myd88. In some instances, the costimulatory domain is modified by amino acid substitutions to modulate their signal transduction qualitatively or quantitatively to induce optimal immune effector cell activity.
5. Intracellular Signaling Domains
[0066] One of skill in the art will recognize intracellular signaling domains for use in CAR constructs and would know to make use of such in the present disclosure. An intracellular signaling domain to be used in the disclosure need not be discovered yet, but given this disclosure, one of skill in the art would understand how to barcode the intracellular signaling domain as described herein such that it could be used in a barcoded CAR construct. In embodiments, the intracellular signaling is an activation domain. In some instances, the intracellular signaling domain is modified by amino acid substitutions to modulate their signal transduction qualitatively or quantitatively to induce optimal immune effector cell activity.
[0067] In embodiments, the intracellular signaling domain is all or part of a domain that is involved in the immune effector cell activation pathway. In specific embodiments the intracellular signaling domain is all or a party of Lek, Fyn, or LAT.
[0068] Exemplary activation domains include DAP10 and CD3(^. In addition to a primary T cell activation signal, such as may be initiated by CD3(^ and/or FcsRIy, besides DAP 10 an additional stimulatory signal for immune effector cell proliferation and effector function following engagement of the chimeric receptor with the target antigen may be utilized. For example, part or all of a human costimulatory receptor for enhanced activation of cells may be utilized that could help improve in vivo persistence and improve the therapeutic success of the adoptive immunotherapy. Embodiments of the disclosure include activation domains that have all or a part of IT AM or ITSM sequences. Examples include all or part of activating domains from molecules such as CD3< DAP10, DAP12, 2B4, CD3g, CD3d, CD3e, CD79a, CD79b, or FcRgamma.
[0069] In exemplary aspects, the activation domain comprises the zeta chain of CD3, which comprises three copies of the Immunoreceptor Tyrosine-based Activation Motif (ITAM). An ITAM generally comprises a Tyr residue separated by two amino acids from a Leu or He. In the case of immune cell receptors, e.g., the T cell receptor and the B cell receptor, the IT AMs occur in multiples (at least two), and each ITAM is separated from another by 6-8 amino acids. B. Construct Generation and Barcoded CAR Construct Libraries
[0070] In embodiments of the disclosure, individual barcoded CAR domains are serially ligated together to form a barcoded CAR construct. Each individual barcoded CAR domain for ligation comprises the DNA of at least the individual CAR domain and the barcode identifying the individual CAR domain. The individual barcoded CAR domain can also comprise restriction sites, selection markers or a combination thereof. Fig. 12 illustrates an example of combinatorial serial assembly of barcoded CAR domains. In embodiments, the CAR domains are ligated together using blunt ligation. In specific embodiments, Type II restriction enzymes are used to create blunt ends (Fig. la).
[0071] In embodiments of the disclosure, each set of domains are pooled with a set of a neighboring domains, each comprising a unique barcode that can identify the specific sequence used in the domain. In embodiments neighboring sets of domains are pooled together, digested and ligated together. The now ligated neighboring sets of domains can then be ligated together with another set of neighboring ligated domains to double the length, or ligated neighboring sets can be ligated with a next single neighbor domain set. In embodiments, ligation products are identified with antibiotic selection. Fig. 12 shows an example ligation strategy where a costimulatory domain is ligated to an activation domain. The costimulatory-activation domain construct is then ligated to a transmembrane construct creating a costimulatory-activation- transmembrane domain construct. The costimulatory-activation-transmembrane domain construct is then ligated to a hinge domain, and this construct is then ligated to an antigen receptor domain creating a full DNA CAR construct. Embodiments of the disclosure include CAR constructs comprising 1, 2, 3 or more stimulatory domains. In some embodiments, only one type of antigen recognition domain is comprised in the set of antigen recognition domains. In some embodiments, only one transmembrane domain is comprised in the set of transmembrane domains. In embodiments, a set of connectors comprises a plurality of types of connectors. In embodiments, a set of costimulatory domains comprises a plurality of types of connector domains. In embodiments, a set of activation domains comprises a plurality of types of activation domains. In embodiments, the plurality is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 60, 70, 80 90, or more types (individual sequences) of domains in a set of domains. [0072] With each ligation, the barcode section of the domain DNA are also ligated together. As shown in Fig. 12, two different sets of domains can be pooled together ([Cosi, C0S2. . . ] + [Acti, Act2, . . .]) where each domain can be identified by and comprises a unique barcode (barcodes shown as 2, 4, 6, 8). When pooled and ligated together, a new set of Cos-Act constructs result which include each combination of the two domain types with each individual domain sequence identifiable by the now combined barcode (2-4; 2-8; 6-4; and, 6-8). Other domains can then be sequentially ligated to the Cos-Act constructs combinatorially, resulting in a library of all or most of the combinations of different domains possible while being able to identify the domains through the use of the combined barcodes.
[0073] Embodiments of the disclosure comprise a method of producing a barcoded library of CAR constructs, wherein the sequence of domains in each construct in the library is identifiable through the full barcode of each CAR construct. Methods of making the library include the steps of barcoding each domain within a CAR construct and sequentially ligating the domains forming a barcoded CAR construct.
[0074] In specific embodiments, a library is formed by receiving a plurality of uniquely barcoded antigen recognition domains; receiving one or more uniquely barcoded connector domains; receiving one or more uniquely barcoded transmembrane domains; receiving a plurality of uniquely barcoded costimulatory domains; receiving a plurality of uniquely barcoded activating domains; ligating together, in sequence, an antigen recognition domain, the connector, the transmembrane domain, the costimulatory domain, the activating domain, such that each resulting CAR construct comprises one scFv domain, one connector, one transmembrane domain, one or more costimulatory domains and one activating domain and wherein each CAR construct comprises a barcode comprising, in order, the unique barcode from each of the scFc domain, the connector, the transmembrane domain, the one or more costimulatory domains and the activating domain.
C. Barcoded CAR constructs in Immune Cells
[0075] In embodiments of the disclosure, the CAR construct libraries are transfected into immune cells, such as T-cells. The barcoded CAR constructs may be delivered to the immune cells for testing by any suitable vector, including by a viral vector or by a non-viral vector. Examples of viral vectors include at least retroviral, lentiviral, adenoviral or adeno-associated viral vectors. Examples of non-viral vectors include at least plasmids, transposons, lipids, nanoparticles and so forth. One of skill in the art would be well-equipped to construct a vector through standard recombinant techniques (see, for example, Sambrook et al., 2001 and Ausubel et al., 1996, both incorporated herein by reference) for the expression of the antigen receptors of the present disclosure.
[0076] It is contemplated that the barcoded CAR construct can be introduced into immune cells as naked DNA or in a suitable vector. Methods of stably transfecting cells by electroporation using naked DNA are known in the art. See, e.g., U.S. Patent No. 6,410,319. Naked DNA generally refers to the DNA encoding a chimeric receptor contained in a plasmid expression vector in proper orientation for expression.
[0077] Alternatively, a viral vector (e.g., a retroviral vector, adenoviral vector, adeno- associated viral vector or lentiviral vector) can be used to introduce the chimeric CAR construct into immune cells. Suitable vectors for use in accordance with the method of the present disclosure are non-replicating in the immune cells. A large number of vectors are known that are based on viruses, where the copy number of the virus maintained in the cell is low enough to maintain the viability of the cell, such as, for example, vectors based on HIV, SV40, EBV, HSV or BPV.
[0078] The sequence of the open reading frame encoding the chimeric receptor can be obtained from a genomic DNA source, a cDNA source or can be synthesized (e.g., via PCR) or combinations thereof. Depending upon the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination thereof as it is found that introns stabilize the mRNA. Also, it may be further advantageous to use endogenous or exogenous non-coding regions to stabilize the mRNA.
[0079] In some embodiments, immune cells comprising a barcoded CAR construct of the present disclosure may be identified in vitro or in vivo by including a marker in the expression vector. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector. Generally, a selection marker is one that confers a property that allows for selection. A positive selection marker is one in which the presence of the marker allows for its selection while a negative selection marker is one in which its presence prevents its selection. An example of a positive selection marker is a drug resistance marker.
[0080] Usually the inclusion of a drug selection marker aids in the cloning and identification of transformants. For example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selection markers. In addition to markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions, other types of markers, including screenable markers such as GFP whose basis is colorimetric analysis, are also contemplated. Alternatively, screenable enzymes as negative selection markers, such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized. One of skill in the art would also know how to employ immunologic markers, possibly in conjunction with FACS analysis. The marker used is not believed to be important so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selection and screenable markers are well known to one of skill in the art.
[0081] Methods of making CARs, expressing them in cells (e.g, T-cells) and utilizing the CAR-expressing T-cells in therapy are known in the art. See, e.g., International Patent Application Publication Nos. WO2014/208760, WO2014/190273, WO2014/186469, WO2014/184143, W02014180306, WO2014/179759, WO2014/153270, U.S. Application Publication Nos. US20140369977, US20140322212, US20140322275, US20140322183, US20140301993, US20140286973, US20140271582, US20140271635, US20140274909, European Application Publication No. 2814846, each of which are incorporated by reference in their entirety.
[0082] Cells that express the barcoded CAR constructs may be of any kind, but in specific embodiments they are immune cells, for example, immune effector cells, such as T cells, NK cells, NKT cells or cell lines derived from the said lineages or engineered to have cytotoxic activity that have been modified to express the barcoded CAR constructs and are therefore not found in nature. In embodiments, immune cells can include gamma-delta T cells, macrophages, neutrophils and iPSC-derived effectors, for example.
[0083] In an exemplary embodiment, a cancer cell line culture can be created using CD 19+ Raji cells and CHLA-255 cells transduced with a GFP construct and sorted to obtain pure populations. For example, Raji and Daudi cells can be maintained in RPMT-10 media (RPML 1640 media with 10% FBS and 1% GlutaMAXTM, ThermoFisher) and CHLA-255 cells in IMDM-20 (IMDM media with 20% FBS and 1% GlutaMAXTM). Cell lines may be routinely STR fingerprinted and tested for mycoplasma contamination. In another exemplary embodiment, Peripheral blood mononuclear cells (PBMCs) can be isolated from buffy coats (Gulf Coast Regional Blood Center) with Ficoll and activated for 48 hours using plate-bound OKT3 and anti- CD28 antibodies (BD Bioscience). Cells can be then resuspended and used for retroviral transduction. T cells can be maintained in RPMI-10 medium supplemented with lOng/ml IL-7 and lOng/ml IL-15, replenished every 48-72 hours.
D. Analysis of Barcoded CAR constructs in CAR T-Cells
[0084] In embodiments, once the barcoded CAR constructs have been transfected into immune cells such as T Cells, the activity of the CAR immune cells are analyzed for activity. In embodiments, CAR T Cells comprising barcoded DNA CAR constructs undergo testing for proliferation, surface expression (i.e. CAR or NGFR), relative expansion, antitumor activity, antivirus activity, antibacterial activity, antifungal activity, removal of other cellular pathogenic states (i.e. senescence, fibrosis, or autoimmunity) or a combination thereof. Such testing can include flow cytometry, cell sorting, activation assays, relative expansion assays, proliferation dye dilution (e.g. CellTrace), measuring cytokine secretion, including IFNy, IL-2, TNFa,in vivo mouse models to test for activity, massively parallel reporter assays, or any combination thereof.
[0085] In embodiments, the CAR immune cells are stimulated prior to testing. For example, the CAR immune cells are stimulated through exposure to an antigen expressing cell, such as a tumor cell, which comprises the antigen to which the antigen recognition domain on the CAR protein binds. In embodiments, the CAR immune cells are exposed to the antigen once or more than once. For example, the CAR immune cells could undergo 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 or 50 cycles of exposure to the antigen.
[0086] In embodiments, the identity of the domains in the CAR construct in the immune cell is identified by barcode comprised on the CAR construct DNA. In embodiments, the barcode is identified by amplification and sequencing. In other embodiments, the barcode is identified by labeled probe hybridization (i.e. fluorescence), in situ sequencing, direct nanopore sequencing or a combination thereof. In embodiments, the barcode is identified before, after or during testing of the barcoded CAR T cells.
[0087] In embodiments, after analysis a barcoded CAR immune cell with high activity compared to other cells in the library is selected. The barcoded CAR immune cell CAR domains are then identified using the barcoded. The specific high activity CAR can then be used to generate immune cells comprising the CAR domains identified in the CAR construct for use in medical treatment. For example, after screening a library of barcoded CAR constructs as described herein, one or more CAR constructs may be identified as performing well to target and kill antigen containing cells (cancer cells, etc.). Once identified, these CARs can be used within CAR T-cell treatments to treat patients in need thereof.
[0088] In embodiments, all or a part of the barcoded CAR construct DNA is sequenced. In embodiments, only the barcode is sequenced. Sequencing the barcoded CAR construct DNA allows the barcode to be identified, thus identifying the sequences of the domains contained thereon. In embodiments, all or a part of the CAR construct is amplified prior to sequencing.
III. Articles of Manufacture or Kits
[0089] An article of manufacture or a kit is provided comprising DNA, proteins, virus, immune cells, antibodies, reagents, buffers or a combination thereof is also provided herein. In embodiments, the kit comprises a DNA barcoded CAR library. In embodiments, the kit comprises the DNA of a plurality of uniquely barcoded antigen recognition domains, one or more uniquely barcoded connector domains, one or more uniquely barcoded transmembrane domains; a plurality of uniquely barcoded costimulatory domains, and a plurality of uniquely barcoded activating domains. The article of manufacture or kit can further comprise a package insert comprising instructions for using the kit to produce barcoded CAR architectures. Any of the methods described herein may be included in the article of manufacture or kits. Suitable containers include, for example, bottles, vials, bags and syringes. The container may be formed from a variety of materials, such as glass, plastic (such as polyvinyl chloride or polyolefin), or metal alloy (such as stainless steel or Hastelloy). In some embodiments, the container holding the formulation and the label on, or associated with, the container may indicate directions for use. The article of manufacture or kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes and package inserts with instructions for use. Suitable containers for the one or more agent include, for example, bottles, vials, bags and syringes.
IV. Sequences used in Certain Embodiments
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
V. Examples
[0090] The following examples are included to demonstrate particular embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
EXAMPLE 1
[0091] Chimeric antigen receptor (CAR)-T cells represent a promising frontier in cancer immunotherapy. However, the current process for developing new CAR constructs is timeconsuming and inefficient. To address this challenge and expedite the evaluation and comparison of full-length CAR designs, we have devised a novel cloning strategy. This strategy involves the sequential assembly of individual CAR domains using blunt ligation with each domain being assigned a unique DNA barcode. Applying this method, we successfully generated 360 CAR constructs that specifically target clinically validated tumor antigens CD 19 and GD2. By quantifying changes in barcode frequencies through next-generation sequencing, we characterize CARs that best mediate proliferation and expansion of transduced T cells. The screening revealed a crucial role for the hinge domain in CAR functionality with CD8a and IgG4 hinges having opposite effects in the surface expression, cytokine production and antitumor activity in CD 19- versus GD2-based CARs. Importantly, we discovered two novel CD19-CAR architectures containing the IgG4 hinge domain that mediate superior in vivo antitumor activity compared to the construct used in Kymriah, an FDA approved therapy. This novel screening approach represents a major advance in CAR engineering enabling accelerated development of cell-based cancer immunotherapies. Using this method, we characterized 360 CAR domain combinations targeting two distinct tumor antigens: CD19 and GD2 (Fig. 13).
[0092] To generate libraries of barcoded CAR domain combinations, we developed an assembly strategy in which individual domains of each class are cloned together through stepwise blunt ligation reactions, and each domain is associated with a single unique barcode (Fig. la). The use of blunt-end ligation reactions enables fusion of protein-coding sequences without altering amino acid sequences at domain junctions. Consequently, each CAR in the library possesses a single predetermined DNA barcode corresponding to each of its serially assembled domains (Fig. la). We generated a CAR library that targets B cell antigen CD 19 using the FMC63 scFv and subcloned it into a retroviral vector with an internal ribosome entry site-nerve growth factor receptor (IRES-NGFR) transduction marker. The library is composed of 180 distinct CAR constructs each uniquely barcoded encompassing various hinge, costimulatory and activating domains (Fig. lb). To validate the accuracy of the assembly process, we sequenced plasmid clones and observed that approximately 80% of the generated CARs had barcodes corresponding to the expected protein domains (Fig. 2a). We transduced this library into Jurkat cells, aiming for a multiplicity of infection (MOI) of 0.3-0.5 to ensure that most cells undergo a single retroviral integration event. Retrovirally-transduced barcoded libraries can theoretically suffer from high recombination rates between constructs, leading to inaccurate barcoding. We performed singlecell sorting of transduced Jurkat cells followed by Sanger sequencing of individual clones and found the barcoding accuracy to be 73% (Fig. 2b). Most discrepancies between expected and sequenced barcodes were found within the costimulatory domain sequences of third-generation CARs. Additionally, there were biases in domain frequencies within the library. For example, the 4-1BB costimulatory domain had a frequency of 2-10% instead of the theoretically expected 20% if the costimulatory domains were evenly distributed (Fig. 2c). Despite these biases, we detected 88-96% of all expected library members (Fig. 2d).
[0093] To assess the feasibility of pooled screening for CAR constructs, we co-cultured 20xl06 CAR-Jurkat cells 1 : 1 with CD19+ Raji cells and assessed cell activation via CD69 immunofluorescence staining (Fig. 14). We isolated CD69-/NGFR+ and CD69+/NGFR+ Jurkat cells via fluorescence-activated cell sorting, then sequenced the DNA barcodes. For each CAR, we calculated the normalized barcode read ratio of CD69+ to CD69- cells revealing that CD69 induction was reproducible between technical replicates (Fig. 1 , R2=0.96). We repeated this experiment using primary T cells and compared CARs containing either the wild-type (wt) or mutant (mt) version of activating domain CD3^ which contains ITAM sequences essential for transforming antigen binding into T cell activation. The ITAM sequences are mutated in CD3qmt to impair T cell activation. As expected, CARs with the CD3 wt domain mediated higher CD69 fold-induction compared to CARs with CD3(^mt (Fig. 16). These results demonstrate that our methodology enables the generation of accurately barcoded libraries of CAR domain combinations that can be used to differentiate between CARs exhibiting expected active versus inactive phenotypes in a pooled screening approach.
[0094] CAR library screening for expansion and proliferation discriminates between active and inactive CARs in primary T cells: We used an intermediate assembly step to create a second library of 180 CARs subcloning the 14g2a scFv that targets neuroblastoma antigen GD2 (Fig. 13). We transduced primary T cells with both CD 19- and GD2-CAR libraries and screened for two clinically relevant phenotypes: relative expansion following four (GD2) or six (CD 19) rounds of antigen stimulation, as well as cell proliferation determined by CellTrace Violet dilution sorting after two rounds of stimulation. Initial testing revealed that T cells transduced with the GD2-CAR library had limited proliferation when co-culturing with tumor cells, but the addition of IL-2 to the co-culture improved this measure. Notably, both CD 19- and GD2-CAR libraries demonstrated significant differences in relative expansion and proliferation between CARs containing the CD3(^wt versus CD3(^mt domain (Figs. 18a-c and 19a-d). Overall, the CD19-CAR library screen identified more active CARs than the GD2-based library, likely due to a combination of scFv- and tumor-specific factors.
[0095] Individual domain analysis reveals hinge domain effect on CAR activity depends on the scFv domain: The combinatorial nature of our screen allowed us to assess how different hinge domains affect the relative expansion and proliferation of T cells expressing CD3 wt CARs. For CD19-CARs, constructs with the IgG4 short hinge mediated higher relative expansion and similar proliferation of transduced T cells compared to constructs with the CD8a hinge (Figs. 20a and 20c). For GD2-CARs, the CD8a hinge outperformed others for both phenotypes (Figs. 20b and 20d). Interestingly, constructs with the CD28 hinge performed the worst in both libraries, suggesting it is a less modular domain likely requiring its corresponding CD28 transmembrane domain for optimal activity. To further compare the IgG4 and CD8a hinges, we generated four CAR constructs with a second-generation 4-1BB costimulatory domain varying the scFv (CD19 or GD2) and hinge domains (IgG4 or CD8a). We first measured individual CAR expression and observed that, compared to IgG4, the CD8a hinge mediated higher levels of CAR surface expression in CD19 CAR-T cells but lower expression in GD2 CAR-T cells (Figs. 20e and 20f). There was no difference in IFNy production for T cells expressing CD19-CARs with either hinge (Fig. 20g). However, T cells expressing the GD2-CAR construct with the CD8a hinge mediated significantly higher IFNy production (Fig. 20h). When examining the impact of the hinges following repeated stimulation, we found no significant impact on tumor control for CD19-CARs regardless of the hinge (Fig. 20i). In contrast, the GD2-CAR demonstrated better tumor control in the construct containing the CD8a hinge versus IgG4, correlating with IFNy production (Fig. 20j). To assess the role of costimulatory domains, we performed a global comparison between second- and third-generation CARs with the CD3 wt domain based on performance in the relative expansion and proliferation screens and found no significant differences between these architectures (Figs. 3a-d). An additional comparison focusing on the membrane-proximal costimulatory domain in each CAR identified 4-1BB as the best performing costimulatory domain; however, this was only statistically significant for the GD2 relative expansion screen (Fig. 4a-d). Unexpectedly, the CD28wt costimulatory domain performed slightly worse than 4-1BB in the proliferation screens contrary to its well-described ability to mediate superior proliferation. This observation could be because CARs with CD28 co-stimulation secrete high levels of cytokines dampening the ability of this domain to induce rapid proliferation in a pooled screening context due to bystander effects. In all, these results suggest that hinge domains impact CAR function based on the scFv domain they are paired with, and they illustrate that evaluating different domain combinations can reveal new functional insights for the overall receptor.
[0096] CAR library screens identify both established and novel domain combinations, with a preference for the 4-1BB costimulatory domain: In addition to a global assessment of domain function, we also evaluated the performance of individual CAR constructs in relative expansion and proliferation screens. In both CD 19- and GD2-CAR screens, we found a correlation between proliferation and relative expansion results (Fig. 21a and 2 Id). Consistent with our previous findings, CD19-CARs with the IgG4 hinge domain mediated more robust relative expansion of transduced cells, while the same was true for the CD8a hinge in GD2-CARs. For CD19-CARs in the relative expansion screen (Fig. 21b), we observed that top hits included CARs with the 4-1BB costimulatory domain alone or with either DAP12 or CD28. We also identified known CAR architectures, including CD8a-41BB, which is the structure of FDA-approved cell therapy for Kymriah CD 19+ malignancies. Interestingly, a construct containing the CD28 hinge domain also performed well on the screens. Comparing CARs between screens, the CD28-41BB-CD28 CAR had better proliferation while IgG4-41BB-DAP12 had better relative expansion (Figs. 21a-c). For GD2, the dominant CARs identified in both screens also had the 4-1BB costimulatory domain alone or with CD28 in third-generation format, as well as the CD8a hinge paired with CD28 (Figs. 21d-f).
[0097] Construct size, costimulatory domain and viral supernatant concentration influence GD2-CAR in vitro antitumor activity: We selected a subset of hits from the GD2 proliferation and relative expansion screens and evaluated the ability of each construct to mediate antitumor activity following repeated stimulation with tumor cells. We found that T cells expressing the CD8a-41BB CAR controlled tumor growth more effectively than those expressing CD8a-OX40-CD28 and CD8a-CD28-41BB, and comparably to CD8a-41BB-CD28 (Fig. 22a). These results show that our screening process identifies both known and novel CAR architectures that mediate potent antitumor activity in transduced T cells. The fact that the GD2-CD8a-41BB CAR mediated effective antitumor activity is contrary to previous observations with this construct using a gammaretroviral LTR expression platform, although this CAR architecture is under clinical investigation. CARs are normally transduced using concentrated supernatant to maximize transduction rates, leading to multi-copy integration. However, our screens were performed using diluted viral supernatant (~3% v/v) leading to an MOI of 0.3-0.5 to maximize single-copy integrations. In addition, the CAR library and individual constructs tested contain an IRES-NGFR sequence, which could also impact transgene expression. We tested GD2 CD8a-41BB CAR constructs with and without the IRES-NGFR sequence and evaluated multiple viral supernatant dilutions for transductions. We found that the transduction rate correlated with CAR surface expression, except at 100% v/v virus concentration where expression of the construct without IRES-NGFR increased out of proportion to the transduction rate (Fig. 22b). The GD2 CD8a-41BB IRES-NGFR construct transduced with 100% v/v viral supernatant was expressed at a similar intermediate level to the construct without IRES-NGFR transduced with lower viral concentrations. This effect was specific for 4-1BB as a GD2-CD8a-CD28 CAR achieved a similar transduction percentage but lower surface expression at high concentrations (Fig. 5).
[0098] Intermediate CAR expression levels led to optimal antitumor activity regardless of the IRES-NGFR sequence while the CAR with the highest surface expression mediated poor persistence and increased exhaustion of transduced cells (Figs. 22C and 22D). To confirm that the improved activity of the IRES-NGFR construct was not sequence-specific, we tested alternative bicistronic vector combinations and found that, despite marginal differences in CAR surface expression, all constructs mediated comparable levels of antitumor activity (Figs. 6a and 6b). In addition, we found that CAR copy number was not significantly lower for the NGFR-IRES construct despite being a larger construct, suggesting an alternative mechanism for the decrease in surface expression at high virus concentrations (Fig. 7). Taken together, these results suggest that our screen was able to identify CAR constructs with optimal expression levels translating to lower levels of exhaustion and higher levels of antitumor activity.
[0099] Screen-selected GD2-CAR constructs mediate comparable levels of in vivo antitumor activity: Next, we evaluated the in vivo antitumor activity of T cells expressing the GD2-CAR with 4-1BB versus CD28 costimulatory domains. We transduced T cells with second- generation CAR constructs containing 4-1BB or CD28 and found that 4-lBB-mediated cell expansion was too low for in vivo testing, in part due to higher apoptosis levels from tonic signaling (Figs. 23a and 23b). To overcome this, we tested different viral concentrations for transduction and found that a 10% v/v viral supernatant improved expansion of cells transduced with the 4-1BB CAR, although not to the same level as mediated by the CD28 CAR (Fig. 23a). Based on this, we compared the 4-1BB CAR transduced at 10% v/v with the CD28 CAR at 100% v/v and infused the cells into NSG mice with metastatic neuroblastoma xenografts. We found that both CD28 and 4-1BB CARs mediated complete elimination of tumor progression in a model with a lower tumor burden while the CD28 CAR mediated superior antitumor activity at a higher tumor dose (Figs. 23c-f). We included an additional group of mice treated with T cells expressing the third generation CAR 4-1BB-CD28, another top construct identified in the GD2 screens. This construct mediated similar levels of antitumor activity to the second generation 4-1BB CAR further supporting the idea that the membrane-proximal costimulatory domain has a dominant effect. [0100] Screen-selected CD19-CAR constructs mediate superior levels of in vivo antitumor activity: The CD 19 screens yielded numerous known and novel CAR structures, and we selected a subset to characterize further. We evaluated CAR expression relative to the NGFR transduction marker in the bicistronic construct used in the screen and found that the CD28-41BB-CD28 CAR is expressed on less than 10% of transduced cells (Fig. 8a) while the other constructs were expressed in over 70% of cells. This supports the idea that the CD28 hinge is less functional in the absence of its transmembrane domain. Consistent with previous findings, we observed that the CD8a hinge mediates higher CAR surface expression compared to IgG4, with third generation CARs having lower surface expression (Fig. 8b). We further characterized these CARs in vitro, first assessing their proliferation as measured by CellTrace Violet dilution (Fig. 24a). Consistent with the pooled screens, the CD28-41BB-CD28 CAR was least proliferative while 41BB-DAP12 was the most, although the differences were not statistically significant. Next, we evaluated in vitro persistence and antitumor activity and found that the IgG4-41BB and IgG4-41BB-CD28 CARs performed similarly to CD8a-41BB (structure of Kymriah) while IgG4-41BB-DAP12 and CD28-41BB-CD28 performed the worst (Figs. 24b-c and Fig. 9). Finally, we measured activation/exhaustion markers but did not find any significant differences between these CARs (Fig. 10). These results suggest that persistence is a more reliable predictor of long-term antitumor activity than short-term proliferation and reinforce that our screen can detect known and novel CARs with therapeutic potential.
[0101] To further assess the therapeutic potential of the top CARs identified in the CD 19 screen, we used an in vivo metastatic Daudi NSG mouse model treating with T cells transduced with IgG4-41BB, IgG4-41BB-CD28 or CD8a-41BB CARs. Surprisingly, both CD19-CARs with the IgG4-41BB architecture mediated better antitumor activity than the Kymriah construct as measured both by tumor bioluminescence and overall survival (Figs. 24d-f). We confirmed this result using CAR T cells from another donor while noticing slight donor-dependent differences between IgG4-41BB and IgG4-41BB-CD28 (Fig. 11). These results suggest that the hinge domain plays a critical role in enhancing in vivo activity consistent with our findings from the pooled screening. EXAMPLE 2
[0102] This work describes a platform for identifying optimized CAR architectures enabled by a novel blunt-end serial barcoded gene assembly strategy. Our CAR assembly method achieves a barcoding accuracy level of 70-80% as described in the previous example. Of note, the barcoding error rate only increased marginally after transducing the libraries, in contrast to prior reports, suggesting that recombination events might be specific to the construct and gene delivery system used. Moreover, we were able to assess up to 96% of expected library members despite having biases in domain distribution, a remarkable improvement over comparable methods.
[0103] Our combinatorial screen shows that CAR domains are not modular and their effects can vary depending on the context. For instance, the presence of the CD28 hinge had a negative impact on CAR surface expression and activity, likely due to lack of its cognate transmembrane domain. Additionally, the effects of the CD8a and IgG4 hinge domains varied depending on the paired scFv. Shorter hinges lead to better activation and cytokine production, although certain antigens might require longer hinges for optimal cell activation and antitumor activity. The hinges included in our libraries in these examples were of similar lengths, indicating that factors other than size can influence hinge function. For example, we found that different scFv-hinge pairings elicit opposite effects on CAR cell surface expression and cytokine secretion. This is likely due to hinge-mediated receptor conformational changes influencing the multivariate activation dynamics downstream of T cell activation.
[0104] The top-performing CAR constructs identified in our GD2 screens featured the 4- 1BB, CD28 and 4-1BB-CD28 costimulatory domains, along with the CD8a hinge. The latter two constructs have already demonstrated some activity in clinical trials, showing the translational potential of this approach. We show that low viral concentrations used for the pooled screening allow for both robust expansion and antitumor activity of cells retrovirally transduced with 4-1BB CARs. Our results suggest there is a threshold level of CAR expression for triggering the deleterious LTR-mediated positive feedback loop. Optimization of CAR expression could improve expansion and antitumor activity of clinical cell therapy products, especially those that initially fail manufacturing. [0105] The second-generation GD2 4-1BB and third-generation 4-1BB-CD28 CARs mediated similar in vivo antitumor activity. This finding aligns with the overall results from our expansion and proliferation screens, where 4-1BB had a dominant effect in the membrane- proximal position, as previously reported for the ICOS costimulatory domain. However, the CD28-containing GD2-CAR mediated the most potent antitumor activity in vivo compared to 4- 1BB despite its relative lower performance in the pooled screens. This may be attributed to the higher levels of cytokine secretion induced by CD28, which is underestimated in pooled screening due to bystander effects. Furthermore, the secretion of cytokines such as IFN-gamma is critical for antitumor activity against solid tumors, which could contribute to the superior in vivo performance mediated by the GD2 CD28 CAR.
[0106] In the case of CD 19, our screening demonstrated that the IgG4 hinge mediated superior in vivo antitumor activity compared to the clinically validated CD8a-41BB CAR architecture, an observation that initially emerged from the screen domain analysis. However, the IgG4 and CD8a hinge-containing CARs were individually compared in vitro, and no differences were detected between them. In some embodiments, pooled screening may be better suited for comparing different CAR architectures in some cases as it may reduce the experimental variability compared with separately manipulating multiple constructs. This could be particularly true in complex, long-term experiments like serial tumor challenges. The hinge domain can also be critical for in vivo activity. For example, the IgG4 hinge with the long CH2-CH3 spacer interacts with myeloid cells, leading to activation-induced T-cell death and poor in vivo persistence.
[0107] In summary, our combinatorial pooled screening approach has generated new insights into CAR structural domains in addition to novel architectures with improved antitumor activity in pre-clinical mouse models compared to known CAR constructs, underscoring the significance of screening full-length CARs. Moreover, systematic mapping of CAR domain interactions, such as optimal scFv-hinge pairs, can offer new design principles for iteratively improving CAR libraries. In some embodiments, this approach enables the study of CAR domain architectures with unprecedented detail and cost-effectiveness; once a combinatorial domain library is generated, it can be used to add any antigen-recognition domain of interest, enabling the rapid development of optimized CARs targeting cancer antigens of interest. EXAMPLE 3
MATERIALS AND METHODS OF PRIOR EXAMPLES
[0108] Construct generation and library assembly: The pSTV28 plasmid (Takara) was modified by the addition of a zeocin selection marker and an N8 DNA barcode primer flanked by Mfel and EcoRI restriction sites, generating pSTV-BC. Domain sub-pools (Table 1) were codon- optimized, restriction sites were removed and DNA fragments were synthesized using gBlocks (IDT). gBlocks were subcloned into pSTV-BC, and domain and barcode sequences were verified by Sanger sequencing, generating for each domain a plasmid with a single, unique barcode, except for CD3(^wt, which had five barcodes. Each domain sub-pool was first digested with either BbsI or Bsal (NEB) and heat-inactivated. Next, sticky ends were blunted with T4 polymerase and dNTPs (NEB), followed by purification using the Monarch PCR & DNA Cleanup Kit (NEB). The product was digested with either Mfel or EcoRI. Then the sub-pool that provides the backbone was processed using Quick CIP (NEB), and the backbone and insert were gel-purified. The insert was ligated into the backbone using T4 Ligase (NEB), then luL was transformed into 10-beta electrocompetent E. coli cells (NEB). Cells were plated onto LB agar plates with ampicillin and zeocin selection, colonies scraped and plasmids extracted with GenElute HP Plasmid Maxiprep Kit (Sigma). These steps were repeated serially until full-length CARs were assembled. To synthesize third-generation CARs, the costimulatory domain was assembled twice. Barcoding accuracy was determined by comparing the CAR coding sequence to the expected barcode via Sanger sequencing of the CAR library from plasmids (A, n=79), single cell Jurkat cell genomic DNA clones PCR (B, n= 43).
[0109] Cancer cell line culture: CD19+ Raji cells (gift from Dr. Gianpietro Doth) and CHLA-255 cells were stably transduced with a GFP construct and sorted to obtain pure populations. Raji and Daudi cells were maintained in RPMI-10 media (RPMI-1640 media with 10% FBS and 1% GlutaMAXTM, ThermoFisher) and CHLA-255 cells in IMDM-20 (IMDM media with 20% FBS and 1% GlutaMAXTM). Cell lines were routinely STR fingerprinted and tested for mycoplasma contamination.
[0110] Primary human T cell isolation and culture: Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats (Gulf Coast Regional Blood Center) with Ficoll and activated for 48 hours using plate-bound 0KT3 and anti-CD28 antibodies (BD Bioscience). Cells were then resuspended and used for retroviral transduction. T cells were maintained in RPMI-10 medium supplemented with lOng/ml IL-7 and lOng/ml IL-15, replenished every 48-72 hours.
[OHl] Retrovirus production and transduction conditions: CAR libraries and individual CAR constructs were subcloned into empty SFG gammaretroviral vectors or SFG modified to include an IRES-NGFR sequence. 293T cells were co-transfected with SFG-CAR and packaging plasmids RD114 and pEQ-PAM3. Retrovirus supernatant was collected after 24 and 48 hours, filtered with a 0.45uM filter and flash frozen in an ethanol-dry ice bath. For transduction, nontissue culture 24-well plates (Falcon) were coated with 7pg/mL Retronectin (Takara) overnight at 4oC. The plates were then washed, retrovirus supernatant was added and plates were centrifuged at 4,000g for 1 hour at room temperature. The supernatant was aspirated, and activated T cells were added at 250,000 cells per well, followed by a 10-minute centrifugation at 1,000g. Transduced cells were expanded for 10 days prior to flow cytometry analysis and tumor co-culture assays.
[0112] Flow cytometry and cell sorting: Virus supernatant titer was assessed by serial dilution, and transduction efficiency was measured by NGFR staining. To assess exhaustion, CAR-T cells were stained with anti-scFv, PD-1 and LAG3 antibodies. Cell death was assessed by Annexin V-PE (ThermoFisher) and DAPI staining. CAR surface expression was detected using anti -idiotype 1A7 for the GD2-CAR and anti-FMC63 (Custoscan) for the CD19-CAR. Samples were acquired on an IntelliCyt iQue Screener and analyzed using FlowJo vlO. Cell sorting was performed on a Sony SH800 sorter.
[0113] CD69 CAR-T cell activation assay: T cells or Jurkat cells were transduced with retroviral supernatant at low MOI (1 :30 dilution), then 20xl06 T cells transduced with the CAR library were co-cultured in a 6-well G-Rex plate (Wilson Wolf) with tumor cells at a 1 :2 ratio (effectortumor). After six (Jurkat) or 24 hours (T), cells were stained with CD69, NGFR and anti- scFv antibodies. CD69+/- cells were sorted on a Sony SH800 sorter.
[0114] Repeat antigen stimulation relative expansion: On day 10 post-transduction, 20x106 T cells transduced with CAR library were co-cultured in 6-well G-Rex plates with irradiated tumor cells 1 :2 (effectortumor) with 200 U/mL IL-2 supplementation every other day. Additional cycles of stimulation were performed with 40x106 irradiated tumor cells every 3-4 days for a total of four cycles for GD2 and six cycles for CD19. For individual CAR evaluations, 500,000 CAR+ T cells were co-cultured with 0.5x106 (Raji) or IxlO6 (CHLA-255) tumor cells (non-irradiated, no IL-2 supplementation), then tumor cells were re-added after each cycle. The remaining tumor cells and CAR+ T cells were assessed by flow cytometry after staining with anti- scFv and DAPI and adding CountBright beads (ThermoFisher).
[0115] CellTrace Violet proliferation screening: At day 10 post-transduction, 20xl06 T cells transduced with CAR library were washed with PBS then resuspended at 106 cells/mL to achieve a 1 : 1000 dilution in CellTrace Violet. Cell suspensions were incubated in the dark for five minutes, RPMI-10 medium was added and suspensions were transferred to a 6-well G-Rex plate. Irradiated tumor cells were added 1 :2 (effectortumor). Four days after adding tumors, a second cycle of stimulation was performed with 40xl06 irradiated tumor cells. Three days later, cells were stained with anti-scFv and NGFR antibodies, and NGFR+ CellTrace Violet-high and -low cells were sorted on a Sony SH800 sorter.
[0116] Library sequencing and analysis: Genomic DNA from CAR-T cell donors was isolated using the DNeasy Blood and Tissue kit (QIAGEN). Quantitative PCR was performed with the KAPA SYBR® FAST qPCR Master Mix (Sigma Aldrich) on a C1000 Touch Thermal Cycler with CFX96 Optical Reaction Module (Bio-Rad) using universal adapter primers. Sample amplification curves were monitored, and PCR was repeated while ensuring that cycle number remained in the exponential phase. Second rounds of both qPCR and PCR were performed using luL of initial PCR product and sample-specific multiplex barcoded primers. Bands were gel- purified using the Monarch DNA Gel Extraction Kit (NEB) and quantified with a Qubit 4 Fluorometer (ThermoFisher). Samples were pooled at equimolar amounts and sequenced using Illumina HiSeq 2xl50bp dual index run averaging ~13 million reads per donor per screen. When correcting sequencing errors, barcode conversions allowed for no more than one mismatch per 8- base barcode. For the initial testing of the CD19 library via CD69 activation, read counts were normalized per sample and ratios of CD69+ to CD69- reads were calculated for each CAR. For repeat antigen stimulation and CellTrace Violet dilution screens, four donor sample pairs for CD 19 and GD2-CAR libraries were compared. DESeq2 was used to normalize reads and calculate foldchange and p-values comparing paired samples. For the CD3^ wild type versus mutant analysis, only CARs with >2,000 normalized reads and the expected scFv domain were analyzed. For the remaining CAR constructs, only full-length, CD3(^ wild type CARs were analyzed. Graphs and group statistical analyses were performed in GraphPad Prism 9.
[0117] IFNy secretion: Culture supernatants were collected after 24 hours of CAR-T cell co-culture with tumor cells, and IFNy was measured using the Human IFN gamma ELISA MAX Deluxe Set (BioLegend) following the manufacturer’s protocol. Absorbance was read in a Spark 10M Multimode Plate Reader (Tecan) at 450 nm wavelength.
[0118] CAR copy number analysis: Genomic DNA from four CAR-T cell donors was isolated using the DNeasy Blood and Tissue kit (QIAGEN) 10 days after transduction. Primers specific for the anti-GD2 14g2a scFv were tested for efficiency via standard dilution, and specificity was determined using non-transduced control genomic DNA (Table 2). Standard curves were generated for both GD2 and RPL32. Quantitative PCR was performed with KAPA S YBR® FAST qPCR Master Mix (Sigma Aldrich) on a C 1000 T ouch Thermal Cycler with CFX96 Optical Reaction Module (Bio-Rad). Absolute copy numbers in samples were calculated using standard dilution curves from PCR products, and CAR copy number was calculated as GD2 copy number / (RPL32 copy number x2).
[0119] In vivo mouse models: For neuroblastoma, NOD.Cg-Prkdcscid I12rgtml Wjl/SzJ (NSG) mice (8- to 10-week-old females, The Jackson Laboratory) were injected intravenously with l-2x!06 CHLA-255 cells engineered to express a FFluc-GFP fusion protein. Two weeks later, mice received a single intravenous injection of 2xl06 CAR-T cells. For CD 19+ lymphoma, mice were injected intravenously with IxlO6 Daudi cells engineered to express a FFluc-GFP fusion protein. Three days later, mice received a single intravenous injection of 2xl06 CAR-T cells. Tumor burden was monitored by recording luminescence on an IVIS Imaging system (Caliper Life Sciences). Mice were euthanized after displaying signs of high tumor burden or >10% weight loss. All animal experiments were conducted in compliance with the Baylor College of Medicine IACUC Protocol AN-5194.
* * *
[0120] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
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The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
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WO2017040694 A2

Claims

WHAT IS CLAIMED IS:
1. A method of producing CAR constructs comprising the steps of: a. receiving a set of uniquely barcoded antigen-recognition domains comprising one or more unique antigen-recognition domains; b. receiving a set of uniquely barcoded connector domains comprising a plurality of unique connector domains; c. receiving a set of uniquely barcoded transmembrane domains comprising one or more unique transmembrane domains; d. receiving a set of uniquely barcoded costimulatory domains comprising a plurality of unique costimulatory domains; e. receiving a set of uniquely barcoded intracellular signaling domains comprising one or more unique intracellular signaling domains; and, f. ligating together the set of uniquely barcoded antigen-recognition domains, the set of uniquely barcoded connector domains, the set of uniquely barcoded transmembrane domains, the set of uniquely barcoded costimulatory domains, and the set of uniquely barcoded costimulatory domains, such that each resulting CAR construct from the ligation comprises, in order and uninterrupted, one or more antigen-recognition domains, one connector domain, one transmembrane domain, one or more costimulatory domains, and one or more intracellular signaling domains and wherein each CAR construct from the ligation comprises a barcode comprising, in order and uninterrupted, the unique barcode from each of the one or more antigen-recognition domains, the unique barcode from the connector domain, the unique barcode from the transmembrane domain, the unique barcode from each of the one or more costimulatory domains, and the unique barcode from each of the one or more intracellular signaling domains.
2. The method of claim 1, additionally comprising the step of transfecting the CAR constructs into immune cells.
3. The method of claim 2, additionally subjecting the immune cells to antigen stimulation.
4. The method of claim 2 or 3, additionally comprising the step of testing the immune cells for proliferation, surface expression, relative expansion, antitumor activity, antivirus activity, antibacterial activity, antifungal activity, removal of cellular pathogenic state or a combination thereof.
5. The method of claim 4, additionally comprising selecting the immune cells with the high antitumor activity, antivirus activity, or antibacterial activity and identifying the CAR construct based on the barcode.
6. The method of claim 5 or 6, additionally comprising generating treatment immune cells comprising the CAR domains identified in the CAR construct.
7. The method of claim 5, additionally comprising administering the treatment immune cells to a patient in need thereof.
8. The method of claim 2, wherein the immune cells are T cells, NK cells, NKT cells, gammadelta T-cells, Macrophages, neutrophils, and iPSC-derived effectors, or a combination thereof.
9. The method of any one of claims 1-8, wherein one of the one or more unique antigen recognition domains binds to CD19, EBNA, CD123, HER2, CA-125, TRAIL/DR4, CD20, CD70, CD38, CD123, CLL1, carcinoembryonic antigen, alphafetoprotein, CD56, AKT, Her3, epithelial tumor antigen, CD319 (CS1), R0R1, folate binding protein, HIV-1 envelope glycoprotein gpl20, HIV-1 envelope glycoprotein gp41, CD5, CD23, CD30, HERV-K, IL-l lRalpha, kappa chain, lambda chain, CSPG4, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD70, TROP-2, CD99, p53, mutated p53, Ras, mutated ras, c-Myc, cytoplasmic serine/threonine kinases (e.g, A-Raf, B-Raf, and C-Raf, cyclin-dependent kinases), MAGE-A1, MAGE-A2, MAGE -A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MART-1, melanoma-associated antigen, BAGE, DAM-6, -10, GAGE-1, -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, MC1R, mda-7, gp75, GplOO, PSA, PSM, Tyrosinase, tyrosinase-related protein, TRP-1, TRP-2, ART -4, CAMEL, CEA, Cyp- B, hTERT, hTRT, iCE, MUC1, MUC2, Phosphoinositide 3-kinases (PI3Ks), TRK receptors, PRAME, P15, RU1, RU2, SART-1, SART-3, Wilms' tumor antigen (WT1), AFP, -catenin/m, Caspase-8/m, CDK-4/m, ELF2M, GnT-V, G250, HAGE, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, Myosin/m, RAGE, S ART-2, TRP- 2/INT2, 707-AP, Annexin II, CDC27/m, TPI/mbcr-abl, BCR-ABL, interferon regulatory factor 4 (IRF4), ETV6/AML, LDLR/FUT, Pml/RAR, Tumor-associated calcium signal transducer 1 (TACSTD1) TACSTD2, receptor tyrosine kinases (e.g, Epidermal Growth Factor receptor (EGFR) (in particular, EGFRvIIT), platelet derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR)), VEGFR2, cytoplasmic tyrosine kinases (e.g., src-family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transducers and activators of transcription STAT3, STATS, and STATE, hypoxia inducible factors (e.g., HIF-1 and HIF-2), Nuclear Factor-Kappa B (NF-B), Notch receptors (e.g., Notch 1-4), NY ESO 1, c-Met, mammalian targets of rapamycin (mTOR), WNT, extracellular signal -regulated kinases (ERKs), and their regulatory subunits, PMSA, PR-3, MDM2, Mesothelin, renal cell carcinoma-5 T4, SM22-alpha, carbonic anhydrases I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL/AML1, GD2, proteinase3, hTERT, sarcoma translocation breakpoints, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin Bl, polysialic acid, MYCN, RhoC, GD3, fucosyl GM1, mesothelian, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SAGE, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNCI, and LRRNl.
10. The method of any one of claims 1-9, wherein one of the one or more unique antigen recognition domains is CD19 FMC63, GD2 14G2a, or a Designed Ankyrin Repeat Protein.
11. The method of any one of claims 1-10, wherein one of the plurality of unique connector domains comprises all or a portion of the hinge domains of CD28, CD8a, CD3^, CD8b, CD4, 41bb, IgG4, IgGl, or IgG2.
12. The method of claim 11, wherein one of the plurality of unique connector domains comprises SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO:5.
13. The method of any one of claims 1-12, one of the one or more unique transmembrane domains comprises all or a portion of the transmembrane domain CD28, CD8a, DAP 10, CD3z, CD8a, CD28, CD8b, 41bb, CD40, CD4, CD3e, CD3g, or CD3d.
14. The method of claim any one of claims 1-12, wherein one of the one or more unique transmembrane domains comprises SEQ ID NO: 6.
15. The method of any one of claims claim 1-14, wherein one of the plurality of unique costimulatory domains comprises all or part of wildtype or mutant signaling domains from one or more of CD28, 41BB or 4-1BB (CD137), ICOS, CD27, CD40, 0X40 (CD134), DAP 12, or Myd88.
16. The method of claim 15, wherein one of the plurality of unique costimulatory domains comprises SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11.
17. The method of any one of claims 1-16, wherein one of the one or more intracellular signaling domains comprises the activating domains from CD3(^, DAP10, DAP12, 2B4, CD3g, CD3d, CD3e, CD79a, CD79b, or FcRgamma; or, wherein one of the one or more unique intracellular signaling domains comprises Lek, Fyn, ZAP70, SLP76 or LAT.
18. The method of claim 17, wherein one of the one or more intracellular signaling domains comprises SEQ ID NO: 12 or SEQ ID NO: 13.
19. The method of any one of claims 1-18, wherein the ligation is blunt end ligation.
20. The method of any one of claims 19, wherein prior to ligation the antigen-recognition domains, the connector domain, the transmembrane domain, the costimulatory domain, the intracellular signaling domain undergo Type Ils digestion.
21. A library of barcoded CAR constructs comprising: a plurality of CAR constructs each comprising, in order and uninterrupted, one or more antigen-recognition domains, one connector domain, one transmembrane domain, one or more costimulatory domains, and one intracellular signaling domain and a unique barcode comprising, in order and uninterrupted, an antigen-recognition domain identifying barcode, a connector domain identifying barcode, a transmembrane domain identifying barcode, one or more costimulatory domain identifying barcodes, and an intracellular signaling domain identifying barcode.
22. The library of claim 21, wherein one of the one or more antigen recognition domain binds to CD19, EBNA, CD123, HER2, CA-125, TRAIL/DR4, CD20, CD70, CD38, CD123, CLL1, carcinoembryonic antigen, alphafetoprotein, CD56, AKT, Her3, epithelial tumor antigen, CD319 (CS1), ROR1, folate binding protein, HIV-1 envelope glycoprotein gpl20, HIV-1 envelope glycoprotein gp41, CD5, CD23, CD30, HERV-K, IL-l lRalpha, kappa chain, lambda chain, CSPG4, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD70, TROP-2, CD99, p53, mutated p53, Ras, mutated ras, c-Myc, cytoplasmic serine/threonine kinases (e.g., A-Raf, B-Raf, and C-Raf, cyclin-dependent kinases), MAGE-A1, MAGE-A2, MAGE- A3, MAGE-A4, MAGE-A6, MAGE- A 10, MAGE-A12, MART-1, melanoma-associated antigen, BAGE, DAM-6, -10, GAGE-1, -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, MC1R, mda-7, gp75, GplOO, PSA, PSM, Tyrosinase, tyrosinase- related protein, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, Phosphoinositide 3-kinases (PI3Ks), TRK receptors, PRAME, P15, RU1, RU2, SART-1, SART-3, Wilms' tumor antigen (WT1), AFP, -catenin/m, Caspase-8/m, CDK-4/m, ELF2M, GnT-V, G250, HAGE, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, Myosin/m, RAGE, S ART-2, TRP-2/INT2, 707-AP, Annexin II, CDC27/m, TPI/mbcr-abl, BCR-ABL, interferon regulatory factor 4 (IRF4), ETV6/AML, LDLR/FUT, Pml/RAR, Tumor-associated calcium signal transducer 1 (TACSTD1) TACSTD2, receptor tyrosine kinases (e.g., Epidermal Growth Factor receptor (EGFR) (in particular, EGFRvIII), platelet derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR)), VEGFR2, cytoplasmic tyrosine kinases (e.g., src-family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transducers and activators of transcription STAT3, STATS, and STATE, hypoxia inducible factors (e.g., HIF-1 and HIF-2), Nuclear Factor-Kappa B (NF-B), Notch receptors (e.g., Notchl-4), NY ESO 1, c-Met, mammalian targets of rapamycin (mTOR), WNT, extracellular signal- regulated kinases (ERKs), and their regulatory subunits, PMSA, PR-3, MDM2, Mesothelin, renal cell carcinoma-5T4, SM22-alpha, carbonic anhydrases I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL/AML1 , GD2, proteinase3, hTERT, sarcoma translocation breakpoints, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin Bl, polysialic acid, MYCN, RhoC, GD3, fucosyl GM1, mesothelian, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SAGE, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE I, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNCI, and LRRNl.
23. The library of claim 21 or 22, wherein one of the one or more antigen recognition domains is CD 19 FMC63, GD2 14G2a, or a Designed Ankyrin Repeat Protein.
24. The library of claim 21, 22, or 23, wherein the connector domain comprises all or a portion of the hinge domains of CD28, CD8a, CD3i CD8b, CD4, 41bb, IgG4, IgGl, or IgG2.
25. The library of claim 24, wherein the connector domain comprises SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.
26. The library of any one of claims 21-25, wherein the transmembrane domain comprises all or a portion of the transmembrane domain CD28, CD8a, DAP 10, CD3z, CD8a, CD28, CD8b, 41bb, CD40, CD4, CD3e, CD3g, or CD3d.
27. The library of any one of claims claim 21-25, wherein the transmembrane domain comprises SEQ ID NO: 6.
28. The library of any one of claims claim 21-27, wherein one of one or more costimulatory domains comprises all or part of wildtype or mutant signaling domains from CD28, 41BB or 4-1BB (CD137), ICOS, CD27, CD40, 0X40 (CD134), DAP12, or Myd88.
29. The library of claim 28, wherein one of the one or more costimulatory domains comprises SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11.
30. The library of any one of claims 21-29, wherein the intracellular signaling domain comprises the activating domains from CD3(^, DAP10, DAP12, 2B4, CD3g, CD3d, CD3e, CD79a, CD79b, or FcRgamma; or, wherein the intracellular signaling domain comprises Lek, Fyn, ZAP70, SLP76 or LAT.
31. The library of claim 30, wherein the intracellular signaling domain comprises SEQ ID NO: 12 or SEQ ID NO: 13.
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