Universal T Cells and the Method of Use Thereof

Abstract

A series of recombinant DNA constructs and a method is disclosed for use in immunological therapy in general; and in disrupting T cell receptor (TCR), human leukocyte antigens (HLA) class I and NKG2D (Natural-Killer Group 2, member D) ligand expression in particular, with the effect of producing highly compatible autologous universal T cells for further genetically engineering for allogeneic administration. A Universal T (UT) construct is provided and used, comprising a TCR antibody fragment fused to a transmembrane domain (TMD) and ER retention domain of adenovirus early region 3 glycoprotein E3-19k (E3/19K) (TCR-E3/19K RD). The Universal T (UT) construct can hijack ERAD machinery to arrest TCR and HLA molecules in endoplasmic reticulum (ER) and facilitate their translocation into the cytoplasm for ubiquitination and degradation by proteasomes.

Claims

1. An amino acid sequence comprising a chimeric UT multi-domain sequence and method of use thereof;

2. The composition of claim 1, wherein the UT multi-domain sequence is composed of a target protein binding moiety, a hinge/linker region, then the transmembrane and cytoplasmic domain of a viral ER-resident glycoprotein;

3. The composition of claim 1, wherein target protein binding moiety could be derived from antibody fragment or natural binding domain, wherein it specifically binds to TCRαβ or HLA molecules;

4. The composition of claim 1, wherein a “hinge region” or a “hinge” refers to (a) an immunoglobulin hinge sequence (made up of, for example, upper and core regions) or a functional fragment or variant thereof, (b) a type II C-lectin interdomain (stalk) region or a functional fragment or variant thereof, or (c) a cluster of differentiation (CD) molecule stalk region or a functional variant thereof. As used herein, a “wild type immunoglobulin hinge region” refers to a naturally occurring upper and middle hinge amino acid sequences interposed between and connecting the CH1 and CH2 domains (for IgG, IgA, and IgD) or interposed between and connecting the CH1 and CH3 domains (for IgE and IgM) found in the heavy chain of an antibody. In certain embodiments, a hinge region is human, and in particular embodiments, comprises a human IgG hinge region. (e.g. IgG hinge; (SEQ ID. NO. 1 to NO.9)); As used herein, a cluster of differentiation (CD) molecule stalk region refers to a natural hinge region derived from the wide type CD molecules, such as CD8, CD4 (e.g. SEQ. NO.10 to NO.14)

5. The composition of claim 1, wherein, in certain embodiments, a connector region may comprise a “linker module” that is an amino acid sequence having from about to two up to about 500 amino acids, which can provide flexibility and room for conformational movement between two regions, domains, motifs, cassettes or modules connected by a linker. Exemplary linker modules include those having from one to about ten repeats of Glyx Sery, wherein x and y are independently an integer from 0 to 10 provided that x and y are not both 0 (e.g., (Gly 4 Ser)2 (SEQ ID NO: 15), (Gly 3Ser)2 (SEQ ID NO:16);

6. The composition of claim 1, wherein the transmembrane domain (TMD) and cytoplasmic domain (CD) of a viral ER-resident glycoprotein, which could hijack ERAD machinery to arrest the binding protein in ER, block its transportation and facilitate its translocation into the cytoplasm for ubiquitination and degradation by proteasomes;

7. The composition of claim 6, wherein the transmembrane domain (TMD) and cytoplasmic domain (CD) of the “viral ER-resident glycoprotein” which is refer to natural virus ER-resident protein, such as HCMV US2, US3, US11, US10, Adenovirus E19, HIV-1 Vpu et al. (e.g. SEQ ID NO:16 to NO:31)

8. The composition of claim 1, wherein the composition the amino acid sequence further comprises with a self-cleavage 2A sequence (e.g. SEQ ID NO:32 to NO.35) and the full length of a viral ER-resident glycoprotein, which could hijack ERAD machinery to suppress MHC-mediated antigen presentation, e.g. HCMV US2, US3, US11, US10, Adenovirus E19 et al. and/or inhibit TAP-mediated peptide transport into the ER and thus block antigen presentation through MHC molecules, e.g. HCMV US6, HSV ICP47.

9. The composition of claim 1 where amino acid sequence further comprises with a self-cleavage 2A sequence (e.g. SEQ ID NO:32 to NO.35) and Chimeric Antigen Receptor (CAR),

10. The composition of claim 9, wherein the CAR includes VH and VL portions of the scFv, which targets TAA, and a hinge, such as a CD8a hinge or IgG4 hinge, attaching the scFc to the transmembrane domain. Intracellular effectors may comprise one or more co-stimulatory signaling domain, such as CD28 intracellular domains (endodomain), 4-1BB(CD137) intracellular domain, fused to CD3ζ.

11. The composition of claim 1 where amino acid sequence further comprises with a self-cleavage 2A sequence (e.g. SEQ ID NO:32 to NO.35) and functional T cell Receptor (TCR), wherein the TCR includes TCR a and b chain fusion protein, coding for a CDR3 region of a TCR recognizing a tumor antigen (TAA).

12. The composition of claim 10, wherein TAA are selected from the groups: (1) Antigens Encoded by Mutated Genes, such as mutated CDK4, CTNNB1, CASP8, P53, KRAS, NRAS, EGFR, EGFRvIII, BRCA1, BRCA2, PALB2, ATM, RAD51D, RECQL, CHEK2, c-MET, or (2) Cancer-Germline Genes, such as melanoma-antigen encoding (MAGE), MAGEA/MAGEB/MAGEC, BAGE, GAGE, LAGE/NY-ESO1, SSX genes, or (3) Differentiation Genes are derived from proteins that are expressed or overexpressed in a given type of tumor and the corresponding healthy tissue, such as tyrosinase, gp100/pmel17, Melan-A/MART-1, gp75/TRP1, TRP2, CEA, CLL1, CCL19, CCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66ae, CD67, CD70, CD70L, CD74, CD79a, CD79b, CD80, CD83, CD95, CD123, CD126, CD132, CD133, CD138, CD147, CD154, gp100, or (4) Overexpressed Antigens contributing to tumor growth or metastasis, such as RAGE-1, PRAME, survivin, ERBB2 (HER2/NEU), protein Wilms tumor 1 (WT1), EpCAM, MUC1(CA15-3), MUC2, MUC3, MUC4, MUC6, MUC16, PMSA, Placental growth factor (PIGF), HIF-1α, EGP-1 (TROP-2), EGP-2, surviving, epidermal glycoprotein 1 (EGP-1, TROP2), EGP-2, FLT3, G250, folate receptor, GAGE, gp100, HLA-DR, CD317(HM1.24), HMGB-1, or (5) Embryonic antigen or fetal antigen or stern cell marker, such as CEA(CEACAM-5), CEACAM-6, AFP, OCT4, CD133, CD90, CD13, c-MET, CDC27, or (6) Tumor metastasis associated chemokine receptor: such as CXCR2, CXCR4, CXCR7, CCR5, CCR7, CCR9, CCR10, CX3CR1 (Lazennec G et al., 2010), or (7) Immune suppressive checkpoint: PD-L1, VISTA, Siglec-15

13. The composition of claim 1 where amino acid sequence further comprises with a self-cleavage 2A sequence (e.g. SEQ ID NO:32 to NO.35) and chimeric NK receptor with adaptor protein fused to T cell activation signaling motif with flexible linker with or without costimulatory domain (See CNK patent US20200308248A1);

14. The composition of claim 1 where the amino acid sequence further comprises with a self-cleavage 2A sequence (e.g. SEQ ID NO:32 to NO.35) and the chemokine receptor, which help to direct T cells toward chemokines expressed by tumors,

15. The composition of claim 14, wherein the chemokine receptors can be CCR4, CCR5, CCR6, CCR7, CCR9, CCR2b, CXCR1, CXCR2, CXCR4 et al,

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1A is a schematic drawing and composition of UT chimeric multi-domains. The wide-type structure of one virus ER-resident glycoprotein (VEP), which is composed of luminal domain (LD), transmembrane domain (TMD) and cytoplasmic functional domain; UT chimeric multi-domain is composed of TCR or HLA binding domain, hinge/spacer, VEP transmembrane and cytoplasmic domain.

[0032] FIG. 1B is a schematic drawing and composition of UT chimeric multi-domains. The wide-type structure of different virus ER-resident glycoproteins (VEP), which is composed of different luminal domain (LD), transmembrane domain (TMD) and cytoplasmic functional domain; UT chimeric multi-domain is composed of TCR or HLA binding domain, hinge/spacer, with different VEP transmembrane and cytoplasmic domain;

[0033] FIG. 1C is a schematic drawing and composition of UT chimeric multi-domains. The wide-type structure of different virus ER-resident glycoproteins (VEP), which is composed of different luminal domain (LD), transmembrane domain (TMD) and cytoplasmic functional domain; UT chimeric multi-domain is composed of TCR or HLA binding domain, hinge/spacer, with the different VEP transmembrane and chimeric cytoplasmic domains derived from different VEP.

[0034] FIG. 2 shows forced expression of UT elements could efficiently suppress TCR expression in Jurkat cells;

[0035] The lentiviral vector with truncated EGFR is encoding anti-TCR scFv fused to different viral ER-resident protein, HCMV US2, US3, US11, Adenovirus E19 TM/CT domain, and the lentiviral vector encoding both anti-TCR scFv fused to both US2 and adenovirus E19 TM/CT were constructed. The Jurkat cells were transduced with such lentivirus and nontransduced Jurkat cells were used as control. 48 h post-transduction, the cells were submitted to flow cytometry to detect TCRαβ expression in the transduced cells (EGFR+ cells). The result shows that all EGFR(+) Jurkat displays significant downregulation of TCRαβ expression; the cells transduced with combined UT composition (US2/E19) could efficiently block TCRαβ expression. All the tested UT chimeric elements (US2, US3, US11, E19) could block TCRαβ expression. Combine UT elements (US2 and E19) could significantly improve the suppression of TCRαβ expression on the Jurkat cell surface.

[0036] FIG. 3A shows forced expression of UT elements could efficiently suppress TCR expression in human T cells. The lentiviral vector with expression cassette including truncated EGFR is encoding anti-TCR scFv fused to HCMV US3 TM/CT domain (anti-TCR-US2 TM/CT-T2A-EGFRt). The human T cells were stimulated and transduced with such lentivirus and the nontransduced T cells as control. 5 days after transduction, the cells were submitted to flow cytometry to detect TCRαβ expression in the transduced T cells (EGFR+ cells). The result shows that EGFR(+) T displays significant downregulation of TCRαβ expression compared to the nontransduced T cells. It indicates US elements containing anti-TCR-US3 TM/CT could efficiently suppress TCRαβ expression in human T cells.

[0037] FIG. 3B shows forced expression of UT elements could efficiently suppress TCR expression in human T cells. The lentiviral vector with expression cassette including truncated EGFR is encoding anti-TCR scFv fused to HCMV E19 TM/CT domain (anti-TCR-E19 TM/CT-T2A-EGFRt). The human T cells were stimulated and transduced with such lentivirus and the nontransduced T cells as control. 5 days after transduction, the cells were submitted to flow cytometry to detect TCRαβ expression in the transduced T cells (EGFR+ cells). The result shows that EGFR(+) T displays significant downregulation of TCRαβ expression compared to the nontransduced T cells. Increased virus transduction could further improve the suppression of TCRαβ expression on the T cell surface. It indicates US elements containing anti-TCR-E19 TM/CT could efficiently TCRαβ expression in human T cells.

[0038] FIG. 4 shows CNK-T cell with UT elements downregulates TCR expression on both CD8/CD4 cells;

[0039] The lentiviral vector contains the expression cassette encoding chimeric NK receptor and anti-TCR scFv fused to E19 TM/CT domain (CNK-UT-E19). The human T cells were transduced with such lentivirus. 5 days post-transduction, the cells were submitted to flow cytometry to detect TCRαβ expression in the transduced cells (NKG2D+ cells), the regular chimeric NK receptor transduced T cells and nontransduced T cells as control. The results show introduction of UT elements into CNK expression cassette will not affect chimeric NK receptor expression. Moreover, it could significantly suppress TCR expression on both CD8+ and CD4+ CNK-T cells.

[0040] FIG. 5 shows CNK-T cell with UT elements display potent cytotoxicity against the tumor cells as the regular CNK-T cells.

[0041] Non-transduced T cells, CNK-T cells and CNK-UT(E19) cells were co-culture with HepG2 cells at E:T ratio=1:5. 24 h post-culture, the cells were harvested and submitted to flow analysis for detect the tumor lysis and T cell activation. The data indicates the CNK-UT cells display comparable potent cytotoxicity against HepG2 cells as regular CNK-T cells. After 24 h co-culture, the CD45(−) tumor cells were significantly decreased in the co-culture with CNK-T as well as CNK-UT. In addition, both CD8 and CD4 CNK-UT cells can upregulate CD25 and CD137 after co-culture with HepG2 cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] The following examples of the invention are included by way of illustration, and not by way of limitation.

[0043] The concept of UT composition is based on the the discovery that virus ER-resident glycoprotein could hijack ERAD tuning machinery to inhibit/block MHC molecules assembly, transportation or promote its ubiquitination and degradation by proteasome, thus suppress MHC-mediated viral antigen presentation for escape of immune surveillance. Efficient downregulation of TCR will significantly suppress the TCR-mediated immune attack and reduce GVHD during allogeneic transfusion of T cells. Including the natural viral ER-resident glycoprotein could further suppress MHC molecules and thereby preventing peptide presentation to recipient CD8+ T cells and suppress immune recognition of allogeneic T cells. Therefore, the UT composition can improve the compatibility and long-term persistence of allogeneic T cells after infusion. For cellular therapeutic purpose, the universal T cells with defective surface expression of TCRαβ and MHC molecules could be further genetically engineered for therapeutic purpose.

Embodiment 1

[0044] UT Elements and Derivatives Thereof

[0045] Exemplary chimeric fusion proteins containing TCRαβ or HLA molecules binding moiety, one or more transmembrane and cytoplasmic domains of viral ER-resident protein in cassettes are illustrated in FIG. 1. For example, in FIG. 1A, the chimeric fusion proteins include the anti-TCRαβ HLA antibody fragment scFv, Hinge/linker (e.g. SEQ NO. 5. IgG4 hinge; and SEQ ID NO.15 (Gly 4 Ser)2 and fused to any transmembrane domain and cytoplasmic domain of ER-resident glycoprotein, e.g. HCMV US2 (SEQ ID NO.19 and NO.20). For example, in FIG. 1B, the chimeric fusion proteins include the anti-TCRαβ or HLA antibody fragment scFv, Hinge/linker (e.g. SEQ NO. 5. IgG4 hinge; and SEQ ID NO.15 (Gly 4 Ser)2 and fused to two or more transmembrane domain and cytoplasmic domain of ER-resident glycoprotein, e.g. HCMV US2 (SEQ ID NO.19 and NO.20) and HCMV US3 (SEQ ID NO.20 and NO.21). For example, in FIG. 1C, the chimeric fusion proteins include the anti-TCRαβ or HLA antibody fragment scFv, Hinge/linker (e.g. SEQ NO. 5. IgG4 hinge; and SEQ ID NO.15 (Gly 4 Ser)2 and fused to the recombinant motif of transmembrane domain and cytoplasmic domain derived from various ER-resident glycoprotein, e.g. HCMV US2 (SEQ ID NO.19 and NO.20) and HCMV US11 (SEQ ID NO.19, NO.20, NO.22, NO.23).

[0046] This UT elements encoding nucleic,' acid molecule was cloned into the lentiviral vector and then transduced into Jurkat cells or human T cells.

Embodiment 2

[0047] Transduced UT elements efficiently block TCRαβ expression in Jurkat cells.

[0048] An exemplary lentiviral construct encoding the UT element comprising anti-TCRαβ fused to the transmembrane and cytoplasmic domain of HCMV US2 or HCMV US11 or/and adenovirus E19, T2A self-cleavage peptide, truncated EGFR was designed (named anti-TCR-US2 TM/CT-T2A-EGFRt; anti-TCR-US11 TM/CT-T2A-EGFRt; anti-TCR-E19 TM/CT-T2A-EGFRt; anti-TCR-US2-TM/CT-T2A-anti-TCR-E19 TM/CT-T2A-EGFRt). The DNA was synthesized and cloned into the lentiviral vector (such as pLenti CMV GFP-puro) and lentivirus were produced in 293T cells, using the package vectors (such as psPAX and pMD2G). Jurkat cells were culture in RPMI, 10% human serum, 2 mM L-glutamine and 1% penicillin-streptomycin (CTL medium) and then transduced with a lentiviral supernatant (as indicated) (MOI=3) supplemented with 0.8 μg/mL polybrene (Millipore, Bedford, Mass.). After 3 days expansion, the cells were submitted to flow cytometry to examine expression of truncated EGFR and TCRαβ in Jurkat cells (FIG. 1). The following conjugated antibodies were used for flow cytometric phenotyping and analysis: CD4-APC, CD8-Pacific Blue, TCR-ab-APC-Cy7, EGFR-PE (Biolegend). Staining with propidium iodide (PI, BD Biosciences) was performed for live/dead cell discrimination as directed by the manufacturer. Flow analyses were done on a FACS Canto II, sort-purifications on a FACS Ariall (Becton Dickinson, Franklin Lakes, N.J.) and data analyzed using FlowJo software (Treestar, Ashland, Oreg.) The result indicated the transduced cells expressed the truncated EGFR and significant downregulation of on TCRαβ on both CD8+ and CD4+ T cells, while the nontransduced Jurkat cells express high level of TCRαβ in absence of truncated EGFR.

Embodiment 3

[0049] Forced expression of UT elements could efficiently suppress TCR expression in human T cells.

[0050] In FIG. 3A, an exemplary lentiviral construct encoding the UT element comprising anti-TCRαβ fused to the transmembrane and cytoplasmic domain of HCMV US2, T2A self-cleavage peptide and truncated EGFR was designed (named anti-TCR-US2 TM/CT-T2A-EGFRt were cloned into the lentiviral vector (such as pLenti CMV GFP-puro) and lentivirus were produced in 293T cells, using the package vectors (such as psPAX and pMD2G). Human CD8+ and CD4+ were isolated from PBMC of normal donors using CD3+ T Cell Isolation Kit (Miltenyi Biotec), activated with anti-CD3/CD28 beads (Life Technologies) according to the manufacturer's instructions, and transduced with a lentiviral supernatant (as indicated in each Example) (MOI=3) supplemented with 0.8 μg/mL polybrene (Millipore, Bedford, Mass.) on day 3 after activation by centrifugation at 2,100 rpm for 45 min at 32° C. T cells were expanded in RPMI, 10% human serum, 2 mM L-glutamine and 1% penicillin-streptomycin (CTL medium), supplemented with recombinant human (rh) IL-2 to a final concentration of 50 U/mL every 48 hours. After 12 days expansion, an aliquot of each transduced T cell line was stained with CD8-Pacific Blue, CD4-APC, TCR-ab-APC-Cy7, EGFR-PE (Biolegend) and then submitted to flow cytometry analysis.

[0051] The result indicates that the transduced T cells (EGFR+) significantly downregulate TCRαβ expression on both CD8+ and CD4+ cell surface compared to the nontransduced T cells. In conclusion, US elements containing anti-TCR-US2 TM/CT could efficiently suppress TCRαβ expression in human T cells which may inhibit TCR-mediated GVHD in vivo after transfusion into different recipient.

[0052] In FIG. 3B, an exemplary lentiviral construct encoding the UT element comprising anti-TCRαβ fused to the transmembrane and cytoplasmic domain of adenovirus E19, T2A self-cleavage peptide and truncated EGFR was designed (named anti-TCR-E19 TM/CT-T2A-EGFRt were cloned into the lentiviral vector (such as pLenti CMV GFP-puro) and lentivirus were produced in 293T cells. Human CD8+ and CD4+ were transduced with two dose of lentiviral supernatant (1×, 2×) and expanded in CTL medium, supplemented with recombinant human (rh) IL-2. After 12 days expansion, an aliquot of each transduced T cell line was stained with CD8-Pacific Blue, CD4-APC, TCR-ab-APC-Cy7, EGFR-PE (Biolegend) and then submitted to flow cytometry analysis.

[0053] The result indicates that the UT(E19) transduced T cells (EGFR+) could also efficiently block TCRαβ expression on both CD8+ and CD4+ cell surface compared to the nontransduced T cells. Increase virus dose of transduction, the suppression of TCRαβ expression could be significantly improved with the increased transduction efficiency and copy number of UT elements in cells. In conclusion, US elements containing anti-TCR-E19 TM/CT could efficiently suppress TCRαβ expression in human T cells which may also inhibit TCR-mediated GVHD in vivo after transfusion into different recipient.

Embodiment 4

[0054] Phenotype of CNK-T Cells Expressing U-T Elements

[0055] In FIG. 4, an exemplary lentiviral construct encoding the CNK sequence, T2A sequence and UT element comprising anti-TCRαβ fused to the TM and CT domain of adenovirus E19, T2A self-cleavage peptide was designed (named CNK-UT-E19 were cloned into the lentiviral vector (such as pLenti CMV GFP-puro) and lentivirus were produced in 293T cells. Human CD8+ and CD4+ were transduced with two dose of lentiviral supernatant and expanded in CTL medium, supplemented with IL-2. After 12 days expansion, an aliquot of each transduced T cell line was stained with CD8-Pacific Blue, CD4-APC, TCR-ab-APC-Cy7, NKG2D-PE-Cy7 (Biolegend) and then submitted to flow cytometry analysis. The results indicate inclusion of UT elements in CNK-T cells could significantly suppress TCRαβ expression in CNK-T cells without interfere with the expression of NKG2D in both CD8+ and CD4+ T cells.

Embodiment 5

[0056] Cytotoxicity of CNK-T Cells Expressing U-T Elements

[0057] The in vitro effector function of CD3+ bulk T cells engineered to express CNK, or CNK-UT were compared to nontransduced T cells respectively—in the co-culture assay with the HCC cell line, HepG2 cells. Briefly, 0.5×10{circumflex over ( )}6 HepG2 cells were seeded on 24 well plates 24 h before the co-culture. The HepG2 cells were added with 1×10{circumflex over ( )}5 effector cells (E:T=1:5) and incubated in the 37 degree for another 24 h before analysis. After co-culture, the cells were harvested and stained with CD8-Pacific Blue, CD4-APC, CD45-PerCP-Cy5.5, CD25-APC-Cy7 and CD137-PE-Cy7 (Biolegend) and then submitted to flow cytometry analysis. The results indicates both CNK-T and CNK-UT(E19) could efficiently eliminate HepG2 in the co-culture assay compared to non-transduced T cells. In addition, CNK-UT cells significantly upregulate CD25 and CD137, the T cell activation markers, as regular CNK-T. In conclusion, inclusion of UT elements will not interfere T cell function, such as cytotoxicity against tumor cells.

[0058] The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments. These and other changes can be made to the embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

TABLE-US-00001 Sequence Listing (SEQ ID NO. 1) = IgG1 hinge: EPKSCDKTHTCPPCP (SEQ ID NO. 2) = IgG2 hinge: ERKCCVECPPCP (SEQ ID NO. 3) = IgG3 hinge: LKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPK SCDTPPPCPRCP (SEQ ID NO. 4) = IgG4 hinge: ESKYGPPCPSCP (SEQ ID NO. 5) = IgG4 hinge(mutated): ESKYGPPCPPCP (SEQ ID NO. 6) = IgA hinge: PVPSTPPTPSPSTPPTPSPSC (SEQ ID NO. 7) = IgD hinge: ESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEE QEERETKTP (SEQ ID NO. 8) = IgM common heavy (CH): IAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQ VGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRG LTFQQNASSMCVPD (SEQ ID NO. 9) = IgE CH2: PTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDL STASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKC A (SEQ ID NO. 10) = CD8a Hinge: AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAA GGAVHTRGLDFACD (SEQ ID NO. 11) = CD8a Hinge: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO. 12) = CD8b Hinge: SVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGPLCSP (SEQ ID NO. 13) = CD8b Hinge: VDFLPTTAQPTKKSTLKKRVCRLPRP ETQKGPLCSP (SEQ ID NO. 14) = CD4 Hinge: DSGQVLLESNIKVLPTWSTPVQP (SEQ ID NO. 15) = (Gly 4 Ser): GGGGS (SEQ ID NO. 16) = (Gly 4 Ser)2: GGGGSGGGGS (SEQ ID NO. 17) = (Gly 3 Ser)2: GGGSGGGS (SEQ ID NO. 18) = HCMV US2 TMD: VAWTIVFYSINITLLVLFIVY (SEQ ID NO. 19) = HCMV US2 CD: VTVDCNLSMMWMRFFVC (SEQ ID NO. 20) = HCMV US3 TMD: TLLVYLFSLVVLVLLTVGVSA (SEQ ID NO. 21) = HCMV US3 CD: RLRFI (SEQ ID NO. 22) = HCMV US11 TMD: YTLMMVAVIQVFWGLYVKGWL (SEQ ID NO. 23) = HCMV US11 CD: HRHFPWMFSDQW (SEQ ID NO. 24) = HCMV US10 TMD: LGDYGAILKIYFGLFCGACVI (SEQ ID NO. 25) = HCMV US10 CD: TRSLLLICGYYPPRE (SEQ ID NO. 26) = HCMV US6 TMD: FFAVTLYLCCGITLLVVILAL (SEQ ID NO. 27) = HCMV US6 CD: LCSITYESTGRGIRRCGS (SEQ ID NO. 28) = AV E19 TMD: TFCSTALLITALALVCTLLYL (SEQ ID NO. 29) = AV E19 CD: KYKSRRSFIDEKKMP (SEQ ID NO. 30) = HIV1 Vpu: TMD: AIVALVVAIIIAIVVWSIVII (SEQ ID NO. 31) = HIV1 CD: EYRKILRQRKIDRLIDRLIERAEDSGNESEGEISALVEMGVEMGHHAPW DVDDL (SEQ ID NO. 32) = T2A: (GSG)EGRGSLLTCGDVEENPGP (SEQ ID NO. 33) = P2A: (GSG)ATNFSLLKQAGDVEENPGP (SEQ ID NO. 34) = E2A: (GSG)QCTNYALLKLAGDVESNPGP (SEQ ID NO. 35) = F2A: (GSG)VKQTLNFDLLKLAGDVESNPGP

[0059] The contents of the following references are incorporated into the disclosure.

[0060] Chevalier, M. S., G. M. Daniels, and D. C. Johnson. 2002. Binding of human cytomegalovirus US2 to major histocompatibility complex class I and II proteins is not sufficient for their degradation. J. Virol. 76:8265-8275.

[0061] Tomazin, R., J. Boname, N. R. Hegde, D. M. Lewinsohn, Y. Altschuler, T. R. Jones, P. Cresswell, J. A. Nelson, S. R. Riddell, and D. C. Johnson. 1999. Cytomegalovirus US2 destroys two components of the MHC class II pathway, preventing recognition by CD4+ T cells. Nat. Med. 5:1039-1043.

[0062] Ben-Arieh, S. V., B. Zimerman, N. I. Smorodinsky, M. Yaacubovicz, C. Schechter, I. Bacik, J. Gibbs, J. R. Bennink, J. W. Yewdell, J. E. Coligan, H. Firat, F. Lemonnier, and R. Ehrlich. 2001. Human cytomegalovirus protein US2 interferes with the expression of human HFE, a nonclassical class I major histocompatibility complex molecule that regulates iron homeostasis. J. Virol. 75:10557-10562.

[0063] Ahn K, Meyer T H, Uebel S, Sempé P, Djaballah H. et al. Molecular mechanism and species specificity of TAP inhibition by herpes simplex virus protein ICP47. EMBO J. 1996b; 15:3247-55.

[0064] York I A, Roop C, Andrew D W, Riddell S R, Graham F L, Johnson D C. A cytosolic herpes simplex virus protein inhibits antigen presentation to CD8+ T lymphocytes. Cell. 1994; 77:525-35.

[0065] Früh K, Ahn K, Djaballah H, Sempé P, van Endert P M. et al. A viral inhibitor of peptide transporters for antigen presentation. Nature. 1995; 375:415-18

[0066] Hill A, Jugovic P, York I, Russ G, Bennink J. et al. Herpes simplex virus turns off the TAP to evade host immunity. Nature. 1995; 375:411-14.

[0067] Furman M H, Dey N, Tortorella D, Ploegh H L. The human cytomegalovirus US10 gene product delays trafficking of major histocompatibility complex class I molecules. J Virol. 2002; 76(22):11753-6.

[0068] Hegde N R, Tomazin R A, Wisner T W, Dunn C, Boname J M, Lewinsohn D M, Johnson D C. Inhibition of HLA-DR assembly, transport, and loading by human cytomegalovirus glycoprotein US3: a novel mechanism for evading major histocompatibility complex class II antigen presentation. J Virol. 2002 November; 76(21):10929-41.

[0069] Chevalier M S, Daniels G M, Johnson D C. Binding of human cytomegalovirus US2 to major histocompatibility complex class I and II proteins is not sufficient for their degradation. J Virol. 2002 August; 76(16):8265-75.

[0070] Chevalier M S, Johnson D C. Human cytomegalovirus US3 chimeras containing US2 cytosolic residues acquire major histocompatibility class I and II protein degradation properties. J Virol. 2003 April; 77(8):4731-8.

[0071] Tomazin R, Boname J, Hegde N R, Lewinsohn D M, Altschuler Y, Jones T R, Cresswell P, Nelson J A, Riddell S R, Johnson D C. Cytomegalovirus US2 destroys two components of the MHC class II pathway, preventing recognition by CD4+ T cells. Nat Med. 1999 September; 5(9):1039-43.

[0072] Hegde N R, Tomazin R A, Wisner T W, Dunn C, Boname J M, Lewinsohn D M, Johnson D C. Inhibition of HLA-DR assembly, transport, and loading by human cytomegalovirus glycoprotein US3: a novel mechanism for evading major histocompatibility complex class II antigen presentation. J Virol. 2002 November; 76(21):10929-41.

[0073] Ahn K, Angulo A, Ghazal P, Peterson P A, Yang Y, Früh K. Human cytomegalovirus inhibits antigen presentation by a sequential multistep process. Proc Natl Acad Sci USA. 1996 Oct. 1; 93(20):10990-5.

[0074] Jones T R, Wiertz E J, Sun L, Fish K N, Nelson J A, Ploegh H L. Human cytomegalovirus US3 impairs transport and maturation of major histocompatibility complex class I heavy chains. Proc Natl Acad Sci USA. 1996 Oct. 15; 93(21):11327-33.

[0075] Ahn K, Angulo A, Ghazal P, Peterson P A, Yang Y, Früh K. Human cytomegalovirus inhibits antigen presentation by a sequential multistep process. Proc Natl Acad Sci USA. 1996 Oct. 1; 93(20):10990-5.

[0076] Jones T R, Wiertz E J, Sun L, Fish K N, Nelson J A, Ploegh H L. Human cytomegalovirus US3 impairs transport and maturation of major histocompatibility complex class I heavy chains. Proc Natl Acad Sci USA. 1996 Oct. 15; 93(21):11327-33.

[0077] Chevalier M S, Johnson D C. Human cytomegalovirus US3 chimeras containing US2 cytosolic residues acquire major histocompatibility class I and II protein degradation properties. J Virol. 2003 April; 77(8):4731-8.

[0078] Chevalier M S, Daniels G M, Johnson D C. Binding of human cytomegalovirus US2 to major histocompatibility complex class I and II proteins is not sufficient for their degradation. J Virol. 2002 August; 76(16):8265-75.

[0079] S. S. Vembar, J. L. Brodsky One step at a time: endoplasmic reticulum-associated degradation Nat. Rev. Mol. Cell Biol., 9 (2008), pp. 944-957

[0080] A. Hershko, A. Ciechanover. The ubiquitin system Annu. Rev. Biochem., 67 (1998), pp. 425-479

[0081] Lybarger, L., X. Wang, M. Harris, and T. H. Hansen. 2005. Viral immune evasion molecules attack the ER peptide-loading complex and exploit ER-associated degradation pathways. Curr. Opin. Immunol. 17:71-78.

[0082] Petersen, J. L., C. R. Morris, and J. C. Solheim. 2003. Virus evasion of MHC class I molecule presentation. J. Immunol. 171:4473-4478.

[0083] Tortorella, D., B. E. Gewurz, M. H. Furman, D. J. Schust, and H. L. Ploegh. 2000. Viral subversion of the immune system. Annu. Rev. Immunol. 18:861-926

[0084] Mocarski, E. S., Jr. 2002. Immunomodulation by cytomegaloviruses: manipulative strategies beyond evasion. Trends Microbiol. 10:332-339.

[0085] Stagg H R, Thomas M, van den Boomen D, Wiertz E J, Drabkin H A, Gemmill R M et al. The TRC8 E3 ligase ubiquitinates MHC class I molecules before dislocation from the ER. J Cell Biol 2009; 186: 685-692S.

[0086] Loureiro J, Lilley B N, Spooner E, Noriega V, Tortorella D, Ploegh H L. Signal peptide peptidase is required for dislocation from the endoplasmic reticulum. Nature. 2006 Jun. 15; 441(7095):894-7.

[0087] Tomazin R, Boname J, Hegde N R, Lewinsohn D M, Altschuler Y, Jones T R, Cresswell P, Nelson J A, Riddell S R, Johnson D C

[0088] Cytomegalovirus US2 destroys two components of the MHC class II pathway, preventing recognition by CD4+ T cells. Nat Med. 1999 September; 5(9):1039-43.

[0089] https://www.nature.com/articles/cmi2014105

[0090] Anne Halenius, Carolin Gerke & Hartmut Hengel Classical and non-classical MHC I molecule manipulation by human cytomegalovirus: so many targets—but how many arrows in the quiver? Cellular & Molecular Immunology volume 12, pages 139-153 (2015)

[0091] Eric W Hewitt The MHC class I antigen presentation pathway: strategies for viral immune evasion. Immunology. 2003 October; 110(2): 163-169.