Patent Application
20230346933
The invention belongs to the field of cellular immunotherapy, in particular to engineered T cells, preparation thereof and use thereof.
With the development of tumor therapy, chimeric antigen receptor-T (CAR-T) immunotherapy has gradually gained a great deal of attention. CAR expressed by CAR-T cells generally contains an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain. Usually CAR-T cells are expanded from autologous T cells transduced with a CAR gene of a patient, and are finally reinfused into said patient. CAR-T cells can efficiently recognize tumor antigens, and elicit specific antitumor immune responses without the limitation of major histocompatibility complex (MHC). Currently, the U.S. FDA has approved two autologous CAR-T cell products, Kymriah from Novartis and YesCAR-Ta from Kate for the treatment of refractory relapsed non Hodgkin's lymphoma and acute lymphoblastic leukemia, respectively. Relevant clinical trials have proven that CAR-T is extremely antitumor potential as a fully personalized live cell drug (Maude S L, Laetsch T W, Buechner J, et al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia[J]. New England Journal of Medicine, 2018, 378(5): 439-448; Park J H, Rivière I, Gonen M, et al. Long-term follow-up of CD19 CAR therapy in acute lymphoblastic leukemia[J]. New England Journal of Medicine, 2018, 378(5): 449-459; Schuster S J, Svoboda J, Chong E A, et al. Chimeric antigen receptor T cells in refractory B-cell lymphomas[J]. New England Journal of Medicine, 2017, 377(26): 2545-2554).
The preparation and reinfusion strategies of the above two CAR-T drugs are to collect the patient's autologous peripheral blood, isolate and obtain T cells, integrate the coding gene of CAR into the genome of T cells through viral vectors, then do expansion culture, and finally reinfuse the CAR-T drugs into the patient. This fully individualized CAR-T cell production not only has a long production cycle and high cost, but also brings many uncertainties to reinfusion therapy. For example, failure of CAR-T cell preparation due to insufficient numbers or malfunctioning of the isolated T cells, rapid disease progression of the patient during cell preparation with loss of the therapeutic window, etc. Therefore, a common expectation in the CAR-T field is that of healthy donor derived off-the-shelf allogeneic CAR-T cells to effectively avoid or address the aforementioned production and treatment-related issues and to greatly reduce production costs.
Regarding the preparation of allogeneic CAR-T cells, one major safety concern first needs to be addressed, namely graft-versus-host disease (GVHD). GVHD is an immune rejection against the recipient's normal tissues and organs induced by the recognition of host allotypic tissue antigens by alloreactive T cells in the graft and is lethal when severe. GVHD is mediated through the T cell receptor (TCR) on the surface of allogeneic T cells. TCR is a characteristic signature on the surface of all mature T cells. TCR binds and assembles intracellularly with multiple CD3 subunits in endoplasmic reticulum (ER) through noncovalent bonds, and finally presents as a TCR-CD3 antigen recognition complex on the cell surface.
As early as 2012, Provasi et al. reported using gene editing technology to try to solve the problem of graft-versus-host reaction of allogeneic T cells (Provasi E, Genovese P, Lombardo A, et al. Editing T cell specificity towards leukemia by zinc finger nucleases and lentiviral gene transfer[J]. Nature medicine, 2012, 18(5): 807). They abrogate GVHD response by specifically knocking out TCR genes in donor T cells through zinc-finger nucleases (ZFN), which results in failure in forming TCR/CD3 complex and inability of T cells to recognize host antigens. Since then, a variety of other novel gene editing technologies, such as transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR) are reported to succeed at the laboratory level (Berdien B, Mock U, Atanackovic D, et al. TALEN-mediated editing of endogenous T-cell receptors facilitates efficient reprogramming of T lymphocytes by lentiviral gene transfer[J]. Gene therapy, 2014, 21(6): 539; Ren J, Zhao Y. Advancing chimeric antigen receptor T cell therapy with CRISPR/Cas9[J]. Protein & cell, 2017, 8(9): 634-643). To date, however, only a very limited number of clinical cases of allogeneic CAR-T have been reported (Qasim W, Zhan H, Samarasinghe S, et al. Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited CAR T cells[J]. Science translational medicine, 2017, 9(374): eaaj2013). The slow progress of gene editing based allogeneic CAR-T clinical research may be mainly due to the safety hazards of gene editing (e.g., off targeting leading to erroneous gene editing), the potential for gene editing to reduce in vivo persistence of T cells, and the hurdles encountered in production preparation (e.g., extensive process steps, greater damage to T cells, etc.).
Non-gene editing based allogeneic CAR-T technologies have similarly gained close attention from industry. This technique was derived from a report in 1995 in which the authors showed that intracellular expression of a specific antibody carrying an endoplasmic reticulum retention signal allows efficient retention of the IL-2 receptor protein within the endoplasmic reticulum with almost no presence on the cell surface, which achieves a loss of function similar to gene editing (Richardson J H, Sodroski J G, Waldmann T A, et al. Phenotypic knockout of the high-affinity human interleukin 2 receptor by intracellular single-chain antibodies against the alpha subunit of the receptor[J]. Proceedings of the National Academy of Sciences, 1995, 92(8): 3137-3141). This technique has recently been borrowed to find polypeptides that can efficiently downregulate the surface TCR/CD3 complex and whether there are universal laws, such as which protein in the TCR/CD3 complex or its epitope is most efficiently recognized by the antibody included in the polypeptide. But the results show that antibodies to different targets, different antibody clones to the same target, different ER retention signals, different linkers, and other factors may affect the activity of these antibody sequences to downregulate the surface TCR/CD3 complex; active antibody sequences in turn lack robust down regulatory activity against different donor T cells (Kamiya T, Wong D, Png Y T, et al. A novel method to generate T-cell receptor-deficient chimeric antigen receptor T cells[J]. Blood advances, 2018, 2(5): 517-528; WO2019032916A1; US20180086831A1).
CAR-T cells with downregulated or absent surface TCR/CD3 complex may also improve the efficacy of current autologous CAR-T cell therapies. It is possible that the TCR/CD3 complex that recognizes the patient's own tumor is present on CAR-T cells prepared from the patient's autologous T cells, and simultaneous activation of the TCR/CD3 complex with CAR is known to trigger exhaustion of CD8+ T cells, thereby affecting the therapeutic efficacy of CAR-T cells (Yang Y, Kohler M E, Chien C D, et al. TCR engagement negatively affects CD8 but not CD4 CAR T cell expansion and leukemic clearance[J]. Science translational medicine, 2017, 9(417): eaag1209).
There is a critical need in the field for a method and cells that can efficiently downregulate the surface TCR/CD3 complex of T cells from different donor sources. There is a critical need in the field for a method and cells that can not only express therapeutically useful proteins but also efficiently downregulate the surface TCR/CD3 complex of T cells from different donor origins.
To this end, the description provides reagents, methods, and cells that efficiently downregulate the TCR/CD3 complex on cell surface.
In the first aspect, the description provides an engineered T cell, wherein the T cell expresses a polypeptide that downregulates the cell surface TCR/CD3 complex, wherein the polypeptide comprises a heavy chain variable region (VH) having an amino acid sequence of SEQ ID No: 1 or more than 95% identical to SEQ ID No: 1, and a light chain variable region (VL) having an amino acid sequence of SEQ ID No: 2 or more than 95% identical to SEQ ID No: 2.
In one or more embodiments, the VH of the polypeptide that downregulates the cell surface TCR/CD3 complex comprises HCDR2: INX1X2X3DVT (wherein X1, X2, X3 are any amino acid) having a sequence as shown in SEQ ID No: 9.
In one or more embodiments, the VH of the polypeptide that downregulates the cell surface TCR/CD3 complex comprises HCDR3: AX4GX5X6YDYDGFVY (wherein X4, X5, X6 are any amino acid) having a sequence as shown in SEQ ID No: 15.
In one or more embodiments, the VL of the polypeptide that downregulates the cell surface TCR/CD3 complex comprises LCDR3: QQWX7X8X9X10X11T (wherein X7, X8, X9, X10, X11 are any amino acid) having a sequence as shown in SEQ ID No: 22.
In one or more embodiments, X1 is proline, threonine, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, alanine, valine, leucine, isoleucine, phenylalanine, methionine or tryptophan.
In one or more embodiments, X2 is tyrosine, glycine, asparagine, glutamine, serine, threonine, cysteine, phenylalanine, tryptophan, histidine, alanine, valine, leucine, isoleucine, proline or methionine.
In one or more embodiments, X3 is asparagine, glycine, glutamine, serine, threonine, tyrosine, cysteine, lysine, arginine, or histidine.
In one or more embodiments, X4 is arginine, lysine, or histidine.
In one or more embodiments, X5 is serine, glycine, asparagine, glutamine, threonine, tyrosine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine or tryptophan.
In one or more embodiments, X6 is tyrosine, glycine, asparagine, glutamine, serine, threonine, cysteine, phenylalanine, tryptophan, histidine, alanine, valine, leucine, isoleucine, proline, methionine, lysine, arginine, aspartic acid or glutamic acid.
In one or more embodiments, X7 is serine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, glycine, asparagine, glutamine, threonine, tyrosine, cysteine, histidine, lysine or arginine.
In one or more embodiments, X8 is serine, glycine, asparagine, glutamine, threonine, tyrosine, cysteine, lysine, arginine, histidine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine or tryptophan.
In one or more embodiments, X9 is asparagine, glycine, glutamine, threonine, tyrosine, cysteine, or serine.
In one or more embodiments, X10 is proline, alanine, leucine, isoleucine, phenylalanine, methionine, or tryptophan.
In one or more embodiments, X11 is leucine, glycine, asparagine, glutamine, serine, tyrosine, cysteine, alanine, isoleucine, proline, phenylalanine, methionine or tryptophan.
In one or more embodiments, LCDR3 is selected from the group consisting of SEQ ID No: 23, SEQ ID No: 24, SEQ ID No: 25, SEQ ID No: 26, SEQ ID No: 27, SEQ ID No: 28, SEQ ID No: 29, SEQ ID No: 30, SEQ ID No: 31, SEQ ID No: 32, SEQ ID No: 33, SEQ ID No: 34.
In one or more embodiments, HCDR2 is selected from the group consisting of SEQ ID No: 10, SEQ ID No: 11, SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14.
In one or more embodiments, HCDR3 is selected from the group consisting of SEQ ID No: 16, SEQ ID No: 17, SEQ ID No: 18, SEQ ID No: 19, SEQ ID No: 20, SEQ ID No: 21.
In one or more embodiments, the amino acid sequence of the heavy chain variable region is selected from the group consisting of: (1) SEQ ID No: 42, (2) a variant of SEQ ID No: 42 that comprises said HCDR2 and/or HCDR3, (3) a sequence more than 95% identical to (1) or (2), and/or, the amino acid sequence of the light chain variable region is selected from the group consisting of: (1) SEQ ID No: 43, (2) a variant of SEQ ID No: 43 that comprises said LCDR3; (3) a sequences more than 95% identical to (1) or (2). In one or more embodiments, HCDR2, HCDR3, and LCDR3 are as described herein.
In one or more embodiments, the polypeptide is a single chain antibody.
In one or more embodiments, the polypeptide that downregulates the cell surface TCR/CD3 complex comprises a localization sequence and optionally a signal peptide.
In one or more embodiments, the localization sequence is selected from the group consisting of endoplasmic reticulum retention sequence, Golgi retention sequence, E3 ubiquitin ligase binding sequence, proteasome localization sequence, or lysosome localization sequence; preferably, the endoplasmic reticulum retention sequence comprises or consists of any one of SEQ ID Nos: 35-40, wherein X is any amino acid.
In one or more embodiments, there is a linker is included between the localization sequence and the antibody, wherein the linker is as shown in SEQ ID No: 41.
In one or more embodiments, the signal peptide is as shown in positions 516-537 of SEQ ID No: 44.
In one or more embodiments, the T cells further express a therapeutic protein; preferably, the therapeutic protein is a chimeric antigen receptor.
In one or more embodiments, the T cells contain an expression frame of the therapeutic protein and an expression frame of a polypeptide that downregulates the cell surface TCR/CD3 complex, or the coding sequence of the therapeutic protein and the coding sequence of the polypeptide that downregulates the cell surface TCR/CD3 complex are in the same expression frame.
In embodiments wherein the coding sequence of the therapeutic protein and the coding sequence of the polypeptide that downregulates the cell surface TCR/CD3 complex are in the same expression frame, a linker sequence is included between the coding sequence of the therapeutic protein and the coding sequence of the polypeptide that downregulates the cell surface TCR/CD3 complex, and the linker sequence enables expression of multiple cistrons on a single vector.
In one or more embodiments, the linker sequence is the coding sequence of 2A peptide.
In one or more embodiments, the 2A peptide comprises F2A, P2A, or T2A peptide.
In one or more embodiments, the chimeric antigen receptor contains, from the N-terminus to the C-terminus: a single chain antibody to a tumor antigen, a hinge region, a transmembrane region, and an intracellular region.
In one or more embodiments, the chimeric antigen receptor specifically binds to one or more of the following tumor antigens: EGFRvIII, mesothelin, GD2, Tn antigen, sTn antigen, Tn-O-glycopeptide, sTn-O-glycopeptide, PSMA, CD97, TAG72, CD44v6, CEA, EPCAM, KIT, IL-13Ra2, leguman, GD3, CD171, IL-11Ra, PSCA, MAD-CT-1, MAD-CT-2, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, folate receptor α, ERBB, Her2/neu, MUC1, EGFR, NCAM, ephrin B2, CAIX, LMP2, sLe, HMWMAA, o-acetyl-GD2, folate receptor β, TEM1/CD248, TEM7R, FAP, legumain, HPV E6 or E7, ML-IAP, CLDN6, TSHR, GPRC5D, ALK, polysialic acid, Fos-associated antigen, neutrophil elastase, TRP-2, CYP1B1, sperm protein 17, β HCG, AFP, thyroglobulin, PLAC1, globoH, RAGE1, MN-CA IX, human telomerase reverse transcriptase, intestinal carboxylesterase, mut hsp 70-2, NA-17, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, NY-ESO-1, GPR20, Ly6k, OR51E2, TARP, GFRα4 and a polypeptide fragment of any of these antigens presented on the MHC, as well as CD5, CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD30, CD34, CD37, CD38, CD40, CD53, CD69, CD72, CD73, CD74, CD75, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD123, CD135, CD138, CD179, CD269, Flt3, ROR1, BCMA, FcRn5, FcRn2, CS-1, CXCR4, CXCR5, CXCR7, IL-7/3R, IL7/4/3R and IL4R.
In one or more embodiments, the hinge region is selected from the group consisting of CD8a hinge region, IgG1 Fc CH2CH3 hinge region, IgD hinge region, CD28 extracellular hinge region, IgG4 Fc CH2CH3 hinge region, and CD4 extracellular hinge region.
In one or more embodiments, the transmembrane region is selected from one or more of CD28 transmembrane region, CD8 transmembrane region, CD3ζ transmembrane region, CD134 transmembrane region, CD137 transmembrane region, ICOS transmembrane region, and DAP10 transmembrane region.
In one or more embodiments, the intracellular region is selected from an intracellular region of one or more of CD28, CD134/OX40, CD137/4-1BB, LCK, ICOS, DAP10, CD3ζ, and Fc310.
In one or more embodiments, the chimeric antigen receptor comprises a signal peptide.
In one or more embodiments, the single chain antibody to the tumor antigen is an anti-CD19 single chain antibody.
In one or more embodiments, the hinge region is CD8a hinge region.
In one or more embodiments, the transmembrane region is CD8 transmembrane region.
In one or more embodiments, the intracellular region comprises a 4-1BB intracellular region and human CD3ζ intracellular region.
In one or more embodiments, the light chain variable region sequence of the anti-CD19 single chain antibody comprises or consists of amino acids 23-129 of SEQ ID No: 44.
In one or more embodiments, the heavy chain variable region sequence of the anti-CD19 single chain antibody comprises or consists of amino acids 148-267 of SEQ ID No: 44.
In one or more embodiments, the sequence of the hinge region comprises or consists of amino acids 268-312 of SEQ ID No: 44.
In one or more embodiments, the sequence of the transmembrane region comprises or consists of amino acids 313-336 of SEQ ID No: 44.
In one or more embodiments, the 4-1BB intracellular region comprises or consists of amino acids 337-378 of SEQ ID No: 44; the CD3ζ intracellular region comprises or consists of amino acids 379-490 of SEQ ID No: 44.
In one or more embodiments, the signal peptide of the chimeric antigen receptor comprises or consists of amino acids 1-22 of SEQ ID No: 44.
In the second aspect, the description provides an engineered T cell, wherein the T cells express a polypeptide that downregulates the TCR/CD3 complex on the cell surface, and the polypeptide comprises: an antibody or an antigen binding fragment thereof, or a mutant having at least 90% sequence identity with the antibody or antigen binding fragment thereof and retaining antigen binding activity thereof; wherein the antibodies comprise a heavy chain variable region and a light chain variable region,
Preferably, X1-X11 are as described herein.
In one or more embodiments, LCDR3 is selected from the group consisting of SEQ ID No: 23, SEQ ID No: 24, SEQ ID No: 25, SEQ ID No: 26, SEQ ID No: 27, SEQ ID No: 28, SEQ ID No: 29, SEQ ID No: 30, SEQ ID No: 31, SEQ ID No: 32, SEQ ID No: 33, SEQ ID No: 34. In one or more embodiments, HCDR2 is selected from the group consisting of SEQ ID No: 10, SEQ ID No: 11, SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14. In one or more embodiments, HCDR3 is selected from the group consisting of SEQ ID No: 16, SEQ ID No: 17, SEQ ID No: 18, SEQ ID No: 19, SEQ ID No: 20, SEQ ID No: 21.
In one or more embodiments, the amino acid sequence of the heavy chain variable region is selected from the group consisting of: (1) SEQ ID No: 42, (2) a variant of SEQ ID No: 42 containing said HCDR2 and/or HCDR3, (3) a sequence more than 95% identical to (1) or (2), and/or the amino acid sequence of the light chain variable region is selected from the group consisting of: (1) SEQ ID No: 43, (2) a variant of SEQ ID No: 43 containing said LCDR3; (3) a sequence more than 95% identical to (1) or (2).
In one or more embodiments, the antibodies are single chain antibodies.
In one or more embodiments, the polypeptide that downregulates the cell surface TCR/CD3 complex comprises a localization sequence and optionally a signal peptide.
In one or more embodiments, the localization sequence is selected from the group consisting of endoplasmic reticulum retention sequence, Golgi retention sequence, E3 ubiquitin ligase binding sequence, proteasome localization sequence, or lysosome localization sequence. Preferably, the endoplasmic reticulum retention sequence comprises or consists of any one of SEQ ID Nos: 35-40, wherein X is any amino acid.
In one or more embodiments, a linker is included between the localization sequence and the antibody, preferably the linker is as shown in SEQ ID No: 41.
In one or more embodiments, the signal peptide is as shown in positions 516-537 of SEQ ID No: 44.
In one or more embodiments, the T cells further express a therapeutic protein, preferably, the therapeutic protein is a chimeric antigen receptor.
In one or more embodiments, the T cells contain an expression frame of the therapeutic protein and an expression frame of a polypeptide that downregulates the cell surface TCR/CD3 complex, or the coding sequence of the therapeutic protein and the coding sequence of the polypeptide that downregulates the cell surface TCR/CD3 complex are in the same expression frame.
In the third aspect, the description also provides an antibody or an antigen binding fragment thereof, wherein the amino acid sequence of HCDR1 of the antibody is GYKFTSYV, the amino acid sequence of HCDR2 is INX1X2X3DVT, the amino acid sequence of HCDR3 is AX4GX5X6YDYDGFVY, the amino acid sequence of LCDR1 is SSVSY, the amino acid sequence of LCDR2 is DTS, the amino acid sequence of LCDR3 is QQWX7X8X9X10X11T; wherein, X1-X11 are as claimed in claim 10, and HCDR2 is not INPYNDVT, HCDR3 is not ARGSYYDYDGFVY, and LCDR3 is not QQWSSNPLT.
In one or more embodiments, LCDR3 is selected from the group consisting of SEQ ID No: 23, SEQ ID No: 24, SEQ ID No: 25, SEQ ID No: 26, SEQ ID No: 27, SEQ ID No: 28, SEQ ID No: 29, SEQ ID No: 30, SEQ ID No: 31, SEQ ID No: 32, SEQ ID No: 33, SEQ ID No: 34. In one or more embodiments, HCDR2 is selected from the group consisting of SEQ ID No: 10, SEQ ID No: 11, SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14. In one or more embodiments, HCDR3 is selected from the group consisting of SEQ ID No: 16, SEQ ID No: 17, SEQ ID No: 18, SEQ ID No: 19, SEQ ID No: 20, SEQ ID No: 21.
In one or more embodiments, the amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID No: 42, but the sequences of its HCDR2 and/or HCDR3 are as shown in the second aspect of the description; the amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID No: 43, but the sequence of its LCDR3 is as shown in the second aspect of the description. Preferably, HCDR2 is as shown in SEQ ID No: 9: INX1X2X3DVT, wherein X1, X2, X3 are any amino acid, HCDR3 is as shown in SEQ ID No: 15: AX4GX5X6YDYDGFVY, wherein X4, X5, X6 are any amino acid, LCDR3 is as shown in SEQ ID No: 22: QQWX7X8X9X10X11T, wherein X7, X8, X9, X10, X11 are any amino acid. In one or more embodiments, X1-X11 are as described elsewhere herein. More preferably, LCDR3 is selected from the group consisting of SEQ ID No: 23, SEQ ID No: 24, SEQ ID No: 25, SEQ ID No: 26, SEQ ID No: 27, SEQ ID No: 28, SEQ ID No: 29, SEQ ID No: 30, SEQ ID No: 31, SEQ ID No: 32, SEQ ID No: 33, SEQ ID No: 34. More preferably, HCDR2 is selected from the group consisting of SEQ ID No: 10, SEQ ID No: 11, SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14. More preferably, HCDR3 is selected from the group consisting of SEQ ID No: 16, SEQ ID No: 17, SEQ ID No: 18, SEQ ID No: 19, SEQ ID No: 20, SEQ ID No: 21.
The description also provides a fusion protein comprising a localization sequence, the polypeptide described herein that downregulates the cell surface TCR/CD3 complex, and, optionally, a signal peptide.
In one or more embodiments, the signal peptide is as shown in positions 516-537 of SEQ ID No: 44.
In one or more embodiments, a linker is included between the localization sequence and a polypeptide that downregulates the cell surface TCR/CD3 complex. The linkers are preferably as shown in SEQ ID No: 41.
In one or more embodiments, the fusion protein is as shown in positions 516-804 or 538-804 of SEQ ID No: 44.
In another aspect, the description provides a nucleic acid molecule selected from the group consisting of: (1) a polynucleotide containing the coding sequence of a therapeutic protein and the coding sequence of a polypeptide described herein that downregulates the cell surface TCR/CD3 complex; (2) the coding sequence of the fusion protein described herein; (3) the coding sequences of the antibodies or their antigen binding fragments described herein; and (4) a complementary sequence or fragment of (1) or (2) or (3). Preferably, the therapeutic protein is a chimeric antigen receptor.
In one or more embodiments, a linker sequence is included between the coding sequence of the therapeutic protein and the coding sequence of a polypeptide that downregulates the cell surface TCR/CD3 complex, such that multiple cistrons are capable of being expressed on a single vector.
In one or more embodiments, the linker sequence is the coding sequence of 2A peptide.
In one or more embodiments, the 2A peptide comprises F2A, P2A, or T2A peptide.
In one or more embodiments, the features of the therapeutic protein and the polypeptide that downregulates the cell surface TCR/CD3 complex are as described herein.
In another aspect, the description provides a nucleic acid construct comprising the nucleic acid molecules described herein.
In one or more embodiments, the nucleic acid construct contains an expression frame of the therapeutic protein and an expression frame of the polypeptide that downregulates the cell surface TCR/CD3 complex; or the nucleic acid construct is an expression frame wherein the coding sequence of the therapeutic protein and the coding sequence of the polypeptide that downregulates the cell surface TCR/CD3 complex are in this expression frame.
In one or more embodiments, the nucleic acid construct is a cloning vector or expression vector.
In another aspect, the description provides a lentivirus containing a nucleic acid molecule, a nucleic acid construct described herein.
In another aspect, the description provides a host cell comprising, expressing and/or secreting an antibody or an antigen binding fragment thereof described herein, or a fusion protein and optionally a therapeutic protein described herein. The host cells comprise the nucleic acid molecules, nucleic acid constructs, and/or lentiviruses described herein.
In another aspect, the description provides a pharmaceutical composition containing the antibody or an antigen binding fragment thereof, a fusion protein and optionally a therapeutic protein, a nucleic acid molecule, a nucleic acid construct, a lentivirus, or a cell described herein.
In another aspect, the description provides use of a polypeptide that downregulates the cell surface TCR/CD3 complex or a coding sequence thereof, antibodies or antigen binding fragments thereof, fusion proteins and optionally therapeutic proteins, nucleic acid molecules, nucleic acid constructs, lentiviruses described herein in the preparation of T cells wherein the cell surface TCR is downregulated, or in the preparation of T cells for cancer therapy. Preferably, said polypeptide that downregulates the cell surface TCR/CD3 complex is as described herein.
It is understood that within the scope of the description, each of the above technical features of the description and those specifically described below (such as in examples) may all be combined with one another, thereby constituting a preferred technical solution.
Engineered T cells with decreased expression of the cell surface TCR/CD3 complex are prepared by expressing a polypeptide that downregulates the cell surface TCR/CD3 complex. In the T cells prepared by the method of the description, the expression level of the cell surface TCR/CD3 complex is at least 50%, preferably at least 90%, more preferably at least 99%, or completely inhibited, as compared with a control that does not express the polypeptide.
Herein, the polypeptide that downregulates the cell surface TCR/CD3 complex comprises antibodies or antigen binding fragments thereof. An “antibody” refers to a polypeptide comprising an immunoglobulin sequence element sufficient to enable its specific binding to a target antigen. As is known in the art, a native intact antibody is a tetramer comprising two identical heavy chain polypeptides and two identical light chain polypeptides. Each heavy chain contains at least four domains: variable domain VH at the amino terminus, constant domain CH1, constant domain CH2, and constant domain CH3 at the carboxy terminus. Each light chain contains two domains: the variable domain VL at the amino terminus and the constant domain CL at the carboxy terminus. Each variable domain contains three “complementarity determining regions”: CDR1, CDR2, and CDR3, and four “framework” regions: FR1, FR2, FR3, and FR4. An “antigen binding fragment” refers to a fragment in an antibody that can specifically bind to a target antigen. Antibodies or antigen binding fragments thereof include Fab fragments, Fab′ fragments, F(ab′)2 fragments, Fd′ fragments, Fd fragments, Fv fragments, disulfide bonded Fv fragments, single chain antibodies (scFv), isolated CDR or CDR groups, polypeptide-Fc fusions, single domain antibodies, camel antibodies, masking antibodies, small module immunodrugs, bifunctional antibodies, nanobodies, Humabody antibodies. Single chain antibodies are antibodies composed of antibody heavy and light chain variable regions linked by a short peptide (linker).
In preferred embodiments, the polypeptide that downregulates the cell surface TCR/CD3 complex is an anti-TCR/CD3 antibody that specifically binds to TCR/CD3, more preferably an anti-TCR/CD3 single chain antibody (scFv). The anti-TCR single chain antibodies herein specifically bind TCR1 and/or TCR2. Preferably, the anti-TCR single chain antibodies herein specifically bind all known or unknown TCRs. In one or more embodiments, the anti-TCR/CD3 antibody is selected from the group consisting of UCHT1, OKT3, CTAB5, CTAB2, CTAB3, or CTAB4. Preferably, anti-TCR/CD3 single chain antibodies are derived from these anti-TCR/CD3 antibodies. Exemplarily, the VL and VH sequences of the UCHT1 scFv contain or consist of SEQ ID Nos: 45 and 46, respectively; the VL and VH sequences of the OKT3 scFv contain or consist of SEQ ID Nos: 47 and 48, respectively. In preferred embodiments, the anti-TCR single chain antibody is derived from CTAB5.
In a preferred embodiment, the anti-TCR antibody comprises the heavy chain variable region having the following HCDRs: HCDR1: GYKFTSYV as shown in SEQ ID No: 3, HCDR2: INX1X2X3DVT as shown in SEQ ID No: 9, wherein X1, X2, X3 are any amino acid, HCDR3: AX4GX5X6YDYDGFVY as shown in SEQ ID No: 15, wherein X4, X5, X6 are any amino acid; the light chain variable region having the following LCDRs: LCDR1: SSVSY as shown in SEQ ID No: 6, LCDR2: DTS as shown in SEQ ID No: 7, LCDR3: QQWX7X8X9X10X11T as shown in SEQ ID No: 22, wherein X7, X8, X9, X10, X11 are any amino acid.
In one or more embodiments, X1 is proline, threonine, glycine, asparagine, glutamine, seine, threonine, tyrosine, cysteine, alanine, valine, leucine, isoleucine, phenylalanine, methionine or tryptophan; X2 is tyrosine, glycine, asparagine, glutamine, serine, threonine, cysteine, phenylalanine, tryptophan, histidine, alanine, valine, leucine, isoleucine, proline or methionine; X3 is asparagine, glycine, glutamine, serine, threonine, tyrosine, cysteine, lysine, arginine, or histidine; X4 is arginine, lysine, or histidine; X5 is serine, glycine, asparagine, glutamine, threonine, tyrosine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine or tryptophan; X6 is tyrosine, glycine, asparagine, glutamine, serine, threonine, cysteine, phenylalanine, tryptophan, histidine, alanine, valine, leucine, isoleucine, proline, methionine, lysine, arginine, aspartic acid or glutamic acid; X7 is serine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, glycine, asparagine, glutamine, threonine, tyrosine, cysteine, histidine, lysine or arginine; X8 is serine, glycine, asparagine, glutamine, threonine, tyrosine, cysteine, lysine, arginine, histidine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine or tryptophan; X9 is asparagine, glycine, glutamine, threonine, tyrosine, cysteine, or serine; X10 is proline, alanine, leucine, isoleucine, phenylalanine, methionine, or tryptophan; X11 is leucine, glycine, asparagine, glutamine, serine, tyrosine, cysteine, alanine, isoleucine, proline, phenylalanine, methionine or tryptophan.
Preferably, LCDR3 is selected from the group consisting of SEQ ID No: 23, SEQ ID No: 24, SEQ ID No: 25, SEQ ID No: 26, SEQ ID No: 27, SEQ ID No: 28, SEQ ID No: 29, SEQ ID No: 30, SEQ ID No: 31, SEQ ID No: 32, SEQ ID No: 33, SEQ ID No: 34. In one or more embodiments, HCDR2 is selected from the group consisting of SEQ ID No: 10, SEQ ID No: 11, SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14. In one or more embodiments, HCDR3 is selected from the group consisting of SEQ ID No: 16, SEQ ID No: 17, SEQ ID No: 18, SEQ ID No: 19, SEQ ID No: 20, SEQ ID No: 21.
In some embodiments, the sequence of the heavy chain variable region of the polypeptide that downregulates the cell surface TCR/CD3 complex comprises or consists of the sequence as shown in SEQ ID No: 42, or a variant of SEQ ID No: 42 that comprises the following HCDRs: HCDR1: GYKFTSYV as shown in SEQ ID No: 3, HCDR2: INX1X2X3DVT as shown in SEQ ID No: 9, wherein X1, X2, X3 are any amino acid, HCDR3: AX4GX5X6YDYDGFVY as shown in SEQ ID No: 15, wherein X4, X5, X6 are any amino acid. In one or more embodiments, X1 is proline, threonine, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, alanine, valine, leucine, isoleucine, phenylalanine, methionine or tryptophan; X2 is tyrosine, glycine, asparagine, glutamine, serine, threonine, cysteine, phenylalanine, tryptophan, histidine, alanine, valine, leucine, isoleucine, proline or methionine; X3 is asparagine, glycine, glutamine, serine, threonine, tyrosine, cysteine, lysine, arginine, or histidine; X4 is arginine, lysine, or histidine; X5 is serine, glycine, asparagine, glutamine, threonine, tyrosine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine or tryptophan; X6 is tyrosine, glycine, asparagine, glutamine, serine, threonine, cysteine, phenylalanine, tryptophan, histidine, alanine, valine, leucine, isoleucine, proline, methionine, lysine, arginine, aspartic acid or glutamic acid.
In one or more embodiments, the sequence of the light chain variable region of the polypeptide that downregulates the cell surface TCR/CD3 complex comprises or consists of the sequence as shown in SEQ ID No: 43 or a variant of SEQ ID No: 43 comprising the following LCDRs: LCDR1: SSVSY as shown in SEQ ID No: 6, LCDR2: DTS as shown in SEQ ID No: 7, LCDR3: QQWX7X8X9X10X11T as shown in SEQ ID No: 22, wherein X7, X8, X9, X10, X11 are any amino acid. In one or more embodiments, X7 is serine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, glycine, asparagine, glutamine, threonine, tyrosine, cysteine, histidine, lysine or arginine; X8 is serine, glycine, asparagine, glutamine, threonine, tyrosine, cysteine, lysine, arginine, histidine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine or tryptophan; X9 is asparagine, glycine, glutamine, threonine, tyrosine, cysteine, or serine; X10 is proline, alanine, leucine, isoleucine, phenylalanine, methionine, or tryptophan; X11 is leucine, glycine, asparagine, glutamine, serine, tyrosine, cysteine, alanine, isoleucine, proline, phenylalanine, methionine or tryptophan.
In some embodiments, the sequence of the heavy chain variable region of the polypeptide that downregulates the cell surface TCR/CD3 complex comprises the sequence as shown in SEQ ID No: 42, but wherein HCDR2 is selected from the group consisting of SEQ ID No: 10, SEQ ID No: 11, SEQ ID No: 12, SEQ ID No: 13, and SEQ ID No: 14, and/or HCDR3 is selected from the group consisting of SEQ ID No: 16, SEQ ID No: 17, SEQ ID No: 18, SEQ ID No: 19, SEQ ID No: 20, SEQ ID No: 21. In one or more embodiments, the sequence of the light chain variable region of the polypeptide that downregulates the cell surface TCR/CD3 complex comprises the sequence as shown in SEQ ID No: 43, but wherein LCDR3 is selected from the group consisting of SEQ ID No: 23, SEQ ID NO: 24, SEQ ID No: 25, SEQ ID No: 26, SEQ ID No: 27, SEQ ID No: 28, SEQ ID No: 29, SEQ ID No: 30, SEQ ID SEQ ID No: 31 SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34.
The light chain variable and heavy chain variable regions of the polypeptide that downregulates the cell surface TCR/CD3 complex described herein can be directly connected or through a linker sequence well known in the art, such as the G- and S-containing linker sequences described previously.
In the present description, the polypeptide that downregulates the cell surface TCR/CD3 complex also include mutants that have at least 70% sequence identity with said antibody (especially the anti-TCR antibody described herein) or its antigen binding fragment and retain the antigen binding activity of said antibody or antigen binding fragment. The mutants include: amino acid sequences that have at least 70%, at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity with the reference sequence and retain the biological activity (such as activating T cells, binding to TCR) of the reference sequence. The sequence identity between two aligned sequences can be calculated with, e.g., BLASTp from NCBI. Mutants also include amino acid sequences that have one or several mutations (insertions, deletions, or substitutions) in the reference amino acid sequence while still retaining the biological activity of that reference sequence. Said several mutations usually refer to 1-50, such as 1-20, 1-10, 1-8, 1-5 or 1-3 mutations. The substitution is preferably a conservative substitution. For scFvs, mutations can occur either within CDR regions (including those described previously) or within FR regions, as long as the biological activity of the reference sequence remains after mutation. For example, in the art, conservative substitutions with amino acids of approaching or similar performance generally do not alter the function of a protein or polypeptide. “Amino acids of approaching or similar performance” include, for example, families of amino acid residues with similar side chains, which include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chain (e.g., threonine, valine, isoleucine) and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Accordingly, the substitution of one or several sites in the present polypeptide with another amino acid residue from the same side chain class will not substantially affect its activity. In some embodiments, the polypeptide that downregulates the cell surface TCR/CD3 complex is a fusion protein, which also comprises a localization sequence that can direct the polypeptide to subcellular structures. Said localization sequences may be located at the N- or C-terminus of the polypeptide. The subcellular structures include, but are not limited to, the Golgi apparatus or endoplasmic reticulum, proteasomes, cell membranes, or lysosomes. The localization sequences include an endoplasmic reticulum retention sequence, a Golgi retention sequence, an E3 ubiquitin ligase binding sequence, a proteasome localization sequence, or a lysosome localization sequence. The localization sequence can be connected directly to its adjacent polypeptide or can be connected by a linker sequence that is well-known in the art, such as the G- and S-containing linker sequences described previously. In one or more embodiments, there is a sequence of SEQ ID No: 41 between the localization sequence and its adjacent polypeptide, such as an antibody or an antigen binding fragment thereof.
The “endoplasmic reticulum retention sequence” or “endoplasmic reticulum retention signal” described herein is a short peptide signal sequence. The endoplasmic reticulum retention sequence has corresponding receptors on the membrane of the Golgi apparatus and can be transported back to the endoplasmic reticulum via reflux vesicles. The endoplasmic reticulum retention sequence applicable to the present description may be an endoplasmic reticulum retention sequence well known in the art. The endoplasmic reticulum retention sequence may be located to the C-terminus of the adjacent polypeptide. The “endoplasmic reticulum retention sequence” or “Golgi retention sequence” includes, but is not limited to, or consists of AEKDEL (SEQ ID No: 35), KDEL (SEQ ID No: 36), KKXX (SEQ ID No: 37), KXD (SEQ ID No: 38), KXE (SEQ ID No: 39) or YQRL (SEQ ID No: 40), wherein X is any amino acid. Proteasome localization sequences are obtained by linking antibody sequences to an E3 ubiquitin ligase targeting domain containing sequence and enabling coexpression of the nucleic acid sequence of E3 ubiquitin ligase protein. E3 ubiquitin ligase proteins bind to the Fc domain of antibodies with high affinity and can recruit ubiquitin-proteosome complexes to degrade molecules (e.g., proteins and peptides) bound to antibodies. E3 ubiquitin ligase protein targeting domain sequences encode amino acid sequences selected from human immunoglobulin G (IgG) constant region (Fc) genes, such as IgG1, IgG2, or IgG4, and are used to form fusion proteins containing antibody and Fc domains. The “proteasome localization sequence” comprises, but is not limited to, PEST motif. The “lysosomal localization sequence” comprises, but is not limited to, KFERQ.
Polypeptides that downregulate the cell surface TCR/CD3 complex may also include signal peptides. The signal peptide can be a membrane protein signal peptide, such as the signal peptide of antibody heavy chain, the signal peptide of antibody light chain, CD8 signal peptide, CD28 signal peptide, and CD4 signal peptide. The amino acid sequence of an exemplary signal peptide may comprise or consist of amino acid residues 516-537 of SEQ ID No: 44.
Therapeutic polypeptides can be further expressed in the engineered T cells. In allogeneic therapy, these T cells effectively avoid graft-versus-host reactions. In the treatment with autologous reinfusion, the T cells prepared by the method of the present description avoid CD8+ T cell exhaustion caused by simultaneous activation of TCR/CD3 complex and CAR, thereby improving therapeutic efficacy.
The “therapeutic protein” or “therapeutic polypeptide” used herein refers to a molecule that exerts a corresponding therapeutic activity after expression in cells (particularly T cells), which may be natural or isolated from a natural environment, or may be a substance of manufacture, including but not limited to: a protein or polypeptide derived from a virus, bacterium, plant, animal, a small molecule active peptide, an antigen or a fragment thereof (e.g., antigenic epitope, antigenic determinant), an antibody or a fragment thereof (e.g., heavy chain, light chain, Fab, Fv, scFv), an antigen receptor (e.g., chimeric antigen receptor CAR), a fusion protein or a polypeptide, etc.
Accordingly, the description also provides a CAR-T cell comprising a chimeric antigen receptor (CAR) targeting a tumor antigen of interest and a polypeptide that downregulates the cell surface TCR/CD3 complex. The description also provides a CAR-T cell comprising a polynucleotide encoding a chimeric antigen receptor (CAR) targeting a tumor antigen of interest and a polynucleotide encoding a polypeptide that downregulates the cell surface TCR/CD3 complex.
Herein, suitable T cells can be various T cells well known in the art, especially those routinely used in cellular immunotherapy, including but not limited to peripheral blood T lymphocytes, cytotoxic killer T cells, helper T cells, suppressor/regulatory T cells, γδT cells, cytokine induced killer cells, and tumor infiltrating lymphocytes, among others, and a mixture of any one or more of the above cells. Herein, CAR-T cells refer to T cells that at least express chimeric antigen receptors.
Herein, the chimeric antigen receptor, with the implications known in the art, is an artificially engineered receptor capable of anchoring specific molecules, such as antibodies, that recognize antigens on the surface of tumor cells to immune cells, such as T cells, allowing immune cells to recognize tumor antigens and kill tumor cells. Chimeric antigen receptors suitable for use herein may be various CARs well known in the art. Typically, CARs contain a polypeptide that binds a tumor antigen, a hinge region, a transmembrane region, and one or more intracellular signaling regions.
Polypeptides suitable for binding tumor antigens may be natural polypeptides or synthetic polypeptides; preferably, the synthetic polypeptide is a single chain antibody or Fab fragment. The “tumor antigen” or “tumor surface antigen” described herein include tumor specific antigens and tumor associated antigens expressed on the surface of tumor cells. Tumor specific antigens are specifically expressed on the surface of tumor cells with no expression in normal tissues. Tumor associated antigens are overexpressed on the surface of tumor cells and lowly expressed in normal tissues. Tumor associated antigens include surface antigens of immune cells, such as CD19; antigens involved in growth and differentiation signals; antigens involved in tumor angiogenesis; and tumor stromal and extracellular matrix antigens of tumor supporting structures.
Herein, tumor antigens of interest include, but are not limited to, solid tumor antigens, myeloid tumor antigens, as well as antigens of non-B cell lineage hematologic tumors. Suitable solid tumor antigens include but are not limited to: EGFRvIII, mesothelin, GD2, Tn antigen, sTn antigen, Tn-O-glycopeptide, sTn-O-glycopeptide, PSMA, CD97, TAG72, CD44v6, CEA, EPCAM, KIT, IL-13Ra2, leguman, GD3, CD171, IL-11Ra, PSCA, MAD-CT-1, MAD-CT-2, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, folate receptor α, ERBB (such as ERBB2), Her2/neu, MUC1, EGFR, NCAM, ephrin B2, CAIX, LMP2, sLe, HMWMAA, o-acetyl-GD2, folate receptor β, TEM1/CD248, TEM7R, FAP, Legumain, HPV E6 or E7, ML-IAP, CLDN6, TSHR, GPRC5D, ALK, polysialic acid, Fos-associated antigen, neutrophil elastase, TRP-2, CYP1B1, sperm protein 17, β HCG, AFP, thyroglobulin, PLAC1, globoH, RAGE1, MN-CA IX, human telomerase reverse transcriptase, intestinal carboxylesterase, mut hsp 70-2, NA-17, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, NY-ESO-1, GPR20, Ly6k, OR51E2, TARP, GFRα4 and a polypeptide fragment of any of these antigens presented on the MHC. Suitable B cell antigens include, but are not limited to CD5, CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD30, CD34, CD37, CD38, CD40, CD53, CD69, CD72, CD73, CD74, CD75, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD123, CD135, CD138, CD179, CD269, Flt3, ROR1, BCMA, FcRn5, FcRn2, CS-1, CXCR4, CXCR5, CXCR7, IL-7/3R, IL7/4/3R and IL4R.
In preferred embodiments, the polypeptide herein that binds a tumor antigen is a single chain antibody that specifically binds to any of the above tumor antigens. Herein, a single chain antibody (scFv) refers to an antibody fragment with the ability to bind antigen, composed of antibody light chain variable (VL) region amino acid sequences and heavy chain variable (VH) region amino acid sequences linked by a hinge. Single chain antibodies of interest may be derived from the antibodies of interest. Antibodies of interest may be human antibodies, including human mouse chimeric and humanized antibodies. Antibodies can be secretory, or membrane anchored, preferably membrane anchored. Herein, specific binding refers to the reaction between an antibody or its antigen binding fragment and the antigen to which it is directed. In some embodiments, an antibody that specifically binds to an antigen (or an antibody specific for an antigen) means that the antibody binds to that antigen with an affinity (KD) of less than approximately 10−5 M, e.g., less than approximately 10−6 M, 10−7 M, 10−8 M, 10−9 M, or 10−10 M or less.
Antibodies to a tumor antigen that are applicable to the description may be derived from single chain antibodies to various tumor antigens well known in the art. Single chain antibodies may contain the heavy and light variable regions of the antibody of interest or consist of heavy and light variable regions and, optionally, a linker. The heavy and light chain variable regions may be linked with a well-known linker. Herein, a linker or hinge is the polypeptide fragment that links between different proteins or polypeptides with the aim of maintaining the attached proteins or polypeptides in their respective spatial conformations in order to maintain the function or activity of the proteins or polypeptides. Exemplary linkers include those containing G and/or S. In general, the linker contains one or more repeated motifs. For example, the motif may be GGGS, GGGGS, SSSSG, GSGSA, and GGSGG. Preferably, these motifs are adjacent in the linker sequence with no amino acid residues inserted between the repeats. The linker sequence may contain 1, 2, 3, 4, or 5 repeated motifs. The length of the linker can be 3-25 amino acid residues, such as 3-15, 5-15, 10-20 amino acid residues. In certain embodiments, the linker sequence is a poly-glycine linker sequence. There is no particular limit to the number of glycines in the linker sequence, which is typically 2-20, e.g., 2-15, 2-10, 2-8. In addition to glycine and serine, the linker may contain other known amino acid residues such as alanine (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), glutamine (Q), and others. The length of the linker is typically 15-20 amino acids. In certain embodiments, the different proteins or polypeptides of the present description are linked by (GGGS)n, wherein n is an integer ranging from 1 to 5. In certain embodiments, the light chain variable and heavy chain variable regions of a single chain antibody of the present description are linked by (GGGS)n, wherein n is an integer ranging from 1 to 5. In certain embodiments, the light chain variable and heavy chain variable regions of a single chain antibody of the present description are linked by GSTSGGGSGGTGGGSS (SEQ ID No: 41).
In embodiments wherein the single chain antibody to a tumor antigen is an anti-CD19 single chain antibody, the tumor antigen of interest is CD19, and the single chain antibody of interest is a single chain antibody that specifically binds to CD19. In one or more embodiments, the anti-CD19 single chain antibody is derived from FMC63. Exemplarily, the light chain variable region sequence of the anti-CD19 single chain antibody comprises or consists of amino acids 23-129 of SEQ ID No: 44; the heavy chain variable region of the anti-CD19 single chain antibody comprises or consists of amino acids 148-267 of SEQ ID No: 44, wherein the heavy and light variable regions are linked by a G- and S-containing linker sequence.
Other parts contained in CAR, such as the hinge region, transmembrane region, and intracellular signaling region can be the hinge region, transmembrane region, and intracellular signaling region routinely used to construct various types of CARs.
In the description, the hinge region is a region between the heavy chain CH1 and CH2 functional regions of immunoglobulin, which is proline rich and does not form a helices, prone to stretch and some distortion, in favor of the complementary binding between the antigen binding site of the antibody and the antigenic epitope. The hinge region suitable for the CARs of the present description is well known in the art. The hinge region suitable for use herein may be selected from CD8a hinge region, IgG1 Fc CH2CH3 hinge region, IgD hinge region, CD28 extracellular hinge region, IgG4 Fc CH2CH3 hinge region, and CD4 extracellular hinge region. In one or more embodiments, the hinge region is human CD8a hinge region. The human CD8α hinge region may be derived from CD8a polypeptide chains well known in the art. Exemplarily, the sequence of human CD8a hinge region comprises or consists of amino acids 268-312 of SEQ ID No: 44.
In the description, the transmembrane region may be selected from one or more of CD28 transmembrane region, CD8 transmembrane region, CD3ζ transmembrane region, CD134 transmembrane region, CD137 transmembrane region, ICOS transmembrane region, and DAP10 transmembrane region. Preferably, the transmembrane region of the chimeric antigen receptor used herein is the CD8 transmembrane region. The amino acid sequence of the exemplary transmembrane region comprises or consists of amino acid residues 313-336 of SEQ ID No: 44.
In the description, the intracellular signaling region can be selected from the intracellular signaling region of any one or more of CD28, CD134/OX40, CD137/4-1BB, LCK, ICOS, DAP10, CD3ζ and Fc310/, preferably 4-1BB intracellular signaling region and CD3ζ intracellular signaling region. The amino acid sequence of the intracellular signaling region exemplified herein is shown as amino acid residues 337-490 of SEQ ID No: 44. In one or more embodiments, the 4-1BB intracellular region comprises or consists of amino acids 337-378 of SEQ ID No: 44; the CD3ζ intracellular region comprises or consists of amino acids 379-490 of SEQ ID No: 44.
Therapeutic proteins such as chimeric antigen receptors may also include signal peptides. Signal peptides are short peptide chains (5-30 amino acids in length) that guide the transfer of newly synthesized proteins into a secretory pathway, which generally refer to the amino acid sequence at the N-terminus of newly synthesized polypeptide chains that serves to direct transmembrane transfer (localization) of proteins. The signal peptide may be a membrane protein signal peptide such as CD8 signal peptide, CD28 signal peptide, and CD4 signal peptide. The amino acid sequence of an exemplary signal peptide may comprise or consist of amino acid residues 1-22 of SEQ ID No: 44.
Thus, in one or more embodiments, the CAR contains, from the N-terminus to the C-terminus, optionally a single chain antibody to a tumor antigen, a hinge region, a transmembrane region, one or more intracellular regions. The amino acid sequence of an exemplary chimeric antigen receptor comprises or consists of amino acid residues 23-490 of SEQ ID No: 44, or comprises or consists of amino acid residues 1-490 of SEQ ID No: 44.
The above portions of a chimeric antigen receptor herein, such as a signal peptide, the light and heavy variable regions of a single chain antibody, a hinge region, a transmembrane region, and an intracellular signal region, may be joined directly to one another or by linker sequences well known in the art, such as the G- and S-containing linker sequences described previously. The therapeutic proteins of the present description, as well as polypeptides that downregulate the cell surface TCR/CD3 complex can be linked directly, or by linker sequences such as the G- and S-containing linker sequences described previously. Alternatively, the therapeutic proteins as well as the polypeptides that downregulate the cell surface TCR/CD3 complex also contain linker sequences, such as 2A peptide, which can express multiple polycistrons on a single vector. As is well known in the art, the 2A peptide is a short peptide that can induce ribosome skipping, including F2A, P2A, T2A peptide, etc. In one or more embodiments, the amino acid sequence of the 2A peptide comprises or consists of amino acids 494-515 of SEQ ID No: 44. The 2A peptides can also be linked to the polypeptides on both sides via conventional G- and S-containing linkers.
The present description comprises polynucleotides encoding polypeptides or proteins that downregulate the cell surface TCR/CD3 complex. The polynucleotides of the present description may be in DNA form or RNA form. A DNA form comprises cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single stranded or double stranded. DNA can be either the coding or noncoding strand. The description also includes degenerate variants of a polynucleotide encoding a polypeptide or protein, i.e., a polynucleotide encoding the same amino acid sequence but differing in nucleotide sequence.
The polynucleotides described herein include sequences that have been changed by codon optimization, as long as the amino acid sequence encoded by the polynucleotide is invariant. Codon optimized sequences may show more suitable expressivity for specific species. Methods for codon optimization of polynucleotide sequences are well known in the art.
The polynucleotides of the description may be a coding sequence of a therapeutic protein such as CAR and a coding sequence of a polypeptide that downregulates the cell surface TCR/CD3 complex, or an expression frame of the therapeutic protein and an expression frame of the protein or polypeptide. In the description, the coding sequence refers to the portion of the nucleic acid sequence that directly determines the amino acid sequence of its protein product (e.g., CAR, single chain antibody, hinge region, transmembrane region, intracellular signaling region, or a polypeptide against downregulated cell surface TCR/CD3 complex, etc.). The boundaries of the coding sequence are usually defined by the ribosome binding site immediately upstream of the open reading frame at the 5′ end of the mRNA (for prokaryotic cells) and the transcription termination sequence immediately downstream of the open reading frame at the 3′ end of the mRNA. Coding sequences may include, but are not limited to, DNA, cDNA, and recombinant nucleic acid sequences. In the description, the expression frame refers to the complete elements required for expression of the gene of interest, including a promoter, a gene coding sequence, and a PolyA tailing signal sequence. The polynucleotides described herein may be independently two nucleic acid molecules containing the coding sequence of a therapeutic protein and that of a polypeptide that downregulates the cell surface TCR/CD3 complex, respectively, for example, the expression frame of a therapeutic protein and the expression frame of the polypeptide, respectively; alternatively, the coding sequence of the therapeutic protein and the coding sequence of the polypeptide that downregulates the cell surface TCR/CD3 complex may be linked with a linker to form one nucleic acid molecule, as if the coding sequence of the therapeutic protein and the coding sequence of the polypeptide are within the same expression frame, or both expression frames may be linked with a suitable linker to form one nucleic acid molecule. In certain embodiments, the polynucleotide of the description is a nucleic acid molecule wherein the coding sequence of the therapeutic protein and the coding sequence of the polypeptide are within the same expression frame, which comprises a promoter, a nucleic acid sequence encoding the therapeutic protein and the polypeptide, and a PolyA tailing signal. In one or more embodiments, the polynucleotide further comprises an optional coding sequence of an endoplasmic reticulum retention sequence.
In certain embodiments, the coding sequence or expression frame is integrated into the genome of a T cell. Thus, in these embodiments, an expression frame comprising encoding the therapeutic proteins and the polypeptides described herein is stably integrated in the genome of the T cells described herein.
In one or more embodiments, the polynucleotides of the description contain the coding sequences of the following sequences: a single chain antibody to a tumor antigen, human CD8a hinge region, human CD8 transmembrane region, 4-1BB intracellular region, human CD3ζ intracellular region, F2A peptide, antibody polypeptide, and endoplasmic reticulum retention sequence. In one or more embodiments, the polynucleotide comprises or consists of the following nucleic acid sequences: (1) a polynucleotide comprising the coding sequence of a therapeutic protein described herein and the coding sequence for a polypeptide that downregulates the cell surface TCR/CD3 complex described herein; (2) the coding sequence of the fusion protein described herein; and (3) a complementary sequence or fragment of (1) or (2). In one or more embodiments, the polynucleotide comprises or consists of SEQ ID No: 51.
The description also relates to a nucleic acid construct containing the polynucleotides described herein, and one or more regulatory sequences operationally linked to such sequences. The polynucleotides described herein can be manipulated in a variety of ways to ensure the expression of polypeptides that downregulate the cell surface TCR/CD3 complex or proteins (therapeutic proteins and/or downregulated polypeptides). Nucleic acid constructs can be manipulated according to the difference or requirements of the expression vector prior to insertion of the nucleic acid construct into a vector. Techniques that utilize recombinant DNA methods to change polynucleotide sequences are known in the art.
Regulatory sequences can be suitable promoter sequences. Promoter sequences are often operationally linked to the coding sequence of the protein to be expressed. A promoter may be any nucleotide sequence showing transcriptional activity in a selected host cell, including mutated, truncated, and hybrid promoters, and may be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to that host cell. Regulatory sequences may also be suitable transcription terminator sequences, sequences recognized by host cells to terminate transcription. The terminator sequence is operationally linked to the 3′ end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell can be used in the present description. Regulatory sequences can also be suitable leader sequences, untranslated regions of mRNAs important for host cell translation. The leader sequence is operationally linked to the 5′ end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell can be used in the present description.
In certain embodiments, the nucleic acid construct is a vector. The vector may be a cloning vector, an expression vector, or a homologous recombination vector. The polynucleotides of the present description can be cloned into many types of vectors, e.g., plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Cloning vectors can be used to provide the coding sequences of the therapeutic protein and the polypeptide of the description, such as one nucleic acid molecule containing the coding sequence of the therapeutic protein and the coding sequence of the polypeptide. Expression vectors can be provided to cells in the form of viral vectors. Expression of the polynucleotide of the present description is generally achieved by operationally linking the polynucleotide of the description to a promoter and incorporating the construct into an expression vector. This vector may be suitable for replicating and integrating eukaryotic cells. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence. Viral vector technology is well known in the art and described, for example, in Sambrook et al (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other manuals in virology and molecular biology. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Homologously recombined vectors are used to integrate the expression frame described herein into the host genome.
In general, suitable vectors contain a replication origin, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers functional in at least one organism. For example, in certain embodiments, the description uses a lentiviral vector containing a replication initiation site, a 3′ LTR, a 5′ LTR, the polynucleotides described herein, and optionally a selectable marker.
An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operationally linked to it. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences can also be used, including, but not limited to, simian virus 40 (SV40) early promoter, mouse mammary carcinoma virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukaemia virus promoter, Epstein Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoter, such as, but not limited to, actin promoter, myosin promoter, heme promoter, and creatine kinase promoter.
To assess the expression of therapeutic proteins, polypeptides, or portions thereof, the expression vectors introduced into cells may also comprise either or both selectable marker genes or reporter genes to facilitate the identification and selection of the expressing cells from the population of cells sought to be transfected or infected by viral vectors. In other respects, selectable markers can be carried over a separate stretch of DNA and used in cotransfection procedures. Both selectable markers and reporter genes can be flanked by appropriate regulatory sequences to enable expression in host cells. Useful selectable markers include, for example, antibiotic resistance genes, such as neo, etc.
Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. After DNA has been introduced into recipient cells, the expression of the reporter gene is assayed at an appropriate time. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or the green luciferase. Suitable expression systems are well known and can be prepared by known techniques or commercially available.
The polynucleotides described herein can usually be obtained by PCR amplification. In particular, primers can be designed according to the nucleotide sequence disclosed herein, especially the open reading frame sequence, and the sequence of interest can be amplified with a commercially available cDNA library, or a cDNA library prepared by conventional methods known to those skilled in the art as a template. When the sequence is long, two or more PCR amplifications are often required before each of the amplified fragments is spliced together in the correct order. Alternatively, the nucleic acid molecules described herein can also be synthesized directly.
Methods for introducing genes into cells and for expressing genes into cells are known in the art. A vector can be readily introduced into a host cell by any method in the art, e.g., mammalian, bacterial, yeast, or insect cells. For example, an expression vector can be transferred into host cells by physical, chemical, or biological means.
Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil in water emulsions, micelles, mixed micelles, and liposomes.
Biological methods for introducing polynucleotides into host cells comprise the use of viral vectors, particularly retroviral vectors, which have become the most widely used method for inserting genes into mammalian such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. Several virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for use in gene delivery systems. Techniques known in the art can be exploited to insert selected genes into vectors and package them into retroviral particles. This recombinant virus can subsequently be isolated and delivered to subject cells in vivo or ex vivo. Many retroviral systems are known in the art. Lentiviruses are a genus under the family Retroviridae. Lentiviral vectors are a more complex class of retroviral vectors. Reagents for lentiviral packaging are well known in the art, such as conventional lentiviral vector systems including pRsv-REV, pMDlg-pRRE, pMD2G, and interference plasmids of interest. In one embodiment, the lentiviral vector pWPXL is used.
Thus, in certain embodiments, the description also provides a lentivirus for activating T cells containing the retroviral vector described herein together with the corresponding packaging genes such as gag, pol, vsvg, and/or rev.
In the description, host cells contain, express, and/or secrete the antibodies and/or fusion proteins described herein and, optionally, therapeutic polypeptides. In the description, when it is said that a cell contains or comprises, expresses, secretes a molecule such as a polypeptide, “contain” means that the molecule is included within or on the surface of the cell; “expression” means that the cell produces the molecule; “secretion” means that the cell secretes the expressed molecule out of the cell. Host cells include T cells ultimately used for disease therapeutic purposes, and a variety of cells, such as E. coli cells, which are used in the process of producing CAR-T cells, for example, to provide the coding sequences of the proteins of the description or to provide the vectors described herein. In certain embodiments, a CAR-T cell stably expressing the polypeptide described herein that downregulates the cell surface TCR/CD3 complex is provided.
T cells suitable for the description may be various types of T cells of various origins. For example, T cells can be derived from PBMCs of healthy individuals. In some embodiments, after obtaining T cells, they may be first stimulated and activated with an appropriate amount (e.g., 30-80 ng/ml, such as 50 ng/ml) of CD3 antibody and then cultured in IL2 medium containing an appropriate amount (e.g., 30-80 IU/ml, such as 50 IU/ml) for further use.
Thus, in certain embodiments, the description provides a genetically modified T cell that contains the polynucleotide described herein, or that contains the lentiviral vector described herein, or that has been infected with the lentivirus described herein, or that is prepared using the methods described herein, or that stably expresses the therapeutic proteins and the polypeptides described herein.
T cells of the present description, such as CAR-T cells, can undergo robust in vivo T cell expansion and persist for extended time at high levels in blood and bone marrow, and form specific memory T cells. Without being limited by any particular theory, T cells of the present description can be differentiated in vivo into a central memory like state after encountering and subsequent eliminating target cells expressing the surrogate antigen.
The description also includes a T-cell culture containing the T cells described herein together with a suitable media. Culture media may be those routinely used in the art for T cell culture.
The description also provides a pharmaceutical composition that comprises the T cells described herein as well as pharmaceutically acceptable excipients. In the description, pharmaceutically acceptable excipients refer to vehicles, diluents and/or excipients that are pharmacologically and/or physiologically compatible with the subject and the active ingredient, including but not limited to: pH modifiers, surfactants, carbohydrates, adjuvants, antioxidants, chelators, ionic strength enhancers and preservatives. More specifically, suitable pharmaceutically acceptable excipients may be those commonly used in the art for the administration of T cells, such as CAR-T cells.
Generally, a therapeutically effective amount of T cells is contained in a pharmaceutical composition. A therapeutically effective amount refers to a dose at which the treatment, prevention, mitigation, and/or mitigation of a disease or condition can be achieved in a subject. The effective amount of treatment can be determined by the age, sex, condition and its severity, and other physical condition of a patient. In the description, a subject or patient usually refers to a mammal, especially a human.
A kit containing the nucleic acid constructs described herein is also provided. The kit may also contain various reagents suitable for transfecting said nucleic acid construct into cells, as well as, optionally, instructions directing a person skilled in the art to transfect said recombinant expression vector into cells.
The description also comprises a cell therapy wherein T cells are genetically modified to express the therapeutic proteins and polypeptides described herein and administrated to an object. For example, the administered CAR-T cells are able to kill the recipient's tumor cells. The antitumor immune response caused by CAR-T cells can be an active or passive immune response. Additionally, CAR mediated immune responses may be part of the steps of adoptive immunotherapy, wherein CAR-T cells induce an immune response specific to the antigen binding moiety in CAR.
Accordingly, diseases amenable to treatment with the CARs, polypeptides, their coding sequences, nucleic acid constructs, expression vectors, viruses, or CAR-T cells of the description are related to the tumor antigen single chain antibody contained in the CARs. Accordingly, the diseases described herein encompass various types of cancer associated with tumor antigens as described previously, including solid tumors such as adenocarcinomas, lung cancer, colon cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, cholangiocarcinomas, gallbladder cancer, esophageal cancer, pancreatic cancer, and prostate cancer, and hematologic neoplasms such as leukemias and lymphomas such as B cell lymphomas, mantle cell lymphomas, acute lymphoblastic leukemias, chronic lymphocytic leukemia, hairy cell leukemia, and acute myeloid leukemia, etc. In embodiments wherein the tumor antigen is CD19, the diseases that can be treated with the CARs comprising the anti-CD19 single chain antibody described herein, polypeptides, their coding sequences, nucleic acid constructs, expression vectors, viruses, or CAR-T cell therapy are preferably CD19 mediated diseases; for example, acute/chronic B-lineage lymphocytic leukemia, non Hodgkin lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, and mantle cell lymphoma, etc.
The T cells of the present description can be administered alone or as a pharmaceutical composition. The cellular or pharmaceutical compositions of the description may be administered in a manner suitable for the treatment (or prevention) of a disease. The number and frequency of administration will be determined by various factors, such as the patient's condition, and the type and severity of the patient's disease. Administration of the compositions may be carried out in any convenient manner, including by injection, infusion, implantation, or transplantation. The compositions described herein may be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous injection, or intraperitoneally. In one embodiment, the T cell composition of the description is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the description is preferably administered by intravenous injection. Compositions of T cells can be injected directly into tumors, lymph nodes, or sites of infection.
In some embodiments of the present description, the T cells of the present description or compositions thereof may be combined with other therapies known in the art. The therapies described include, but are not limited to, chemotherapy, radiotherapy and immunosuppressive agents. For example, treatments may be carried out in combination with radiotherapy or chemotherapy formulations that are well known in the art to treat tumor antigen mediated diseases.
In the description, “anti-tumor effect” refers to a biological effect that can be represented by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with cancer.
The gene sequence of an anti-CD19 antibody (specifically, an scFv derived from clone No. FMC63) is used as an example, the sequence information of human CD8a hinge region, human CD8 transmembrane region, 4-1BB intracellular region, human CD3ζ intracellular region and the anti-TCR single chain antibody, etc, is searched from the NCBI GenBank database, and a gene fragment of a whole gene synthetic chimeric antigen receptor is inserted into a lentiviral vector. The recombinant plasmid packages the virus in 293T cells, infects T cells, and allows the T cells to express the chimeric antigen receptor. The present method of transformation to achieve chimeric antigen receptor gene modified T lymphocytes is based on a lentiviral transformation method. This method has the advantages of high conversion efficiency, stable expression of foreign genes, and can shorten the time for in vitro culture of T lymphocytes to reach a clinical grade number. On the surface of this transgenic T lymphocyte, the transformed nucleic acids are expressed by transcription and translation. CAR-T cells prepared by the present description have strong killing function against specific tumor cells, and the killing efficiency of CAR-T cells containing different anti-TCR clone single chain antibodies are all over 98% with an effector-target ratio of 2.5:1.
1. An engineered T cell, wherein the T cell expresses a polypeptide that downregulates a cell surface TCR/CD3 complex, the polypeptide comprises a heavy chain variable region (VH) that has an amino acid sequence of SEQ ID No: 1 or more than 95% identical to SEQ ID No: 1, and a light chain variable region (VL) that has an amino acid sequence of SEQ ID No: 2 or more than 95% identical to SEQ ID No: 2.
2. The T cell as described in embodiment 1, wherein
3. The T cell as described in embodiment 2, wherein the polypeptide that downregulates the cell surface TCR/CD3 complex comprises has one or more features selected from the group consisting of:
4. The T cell as described in any of embodiments 1-3, wherein the polypeptide that downregulates the cell surface TCR/CD3 complex comprises one or more of each of the following groups:
5. The T cell as described in any of embodiments 1-4, wherein the polypeptide is a single chain antibody.
6. The T cell as described in any of embodiments 1-5, wherein the polypeptide that downregulates the cell surface TCR/CD3 complex comprises a localization sequence and optionally a signal peptide, preferably,
7. The T cell as described in any of embodiments 1-6 further expresses a therapeutic protein, preferably the therapeutic protein is a chimeric antigen receptor.
8. The T cell as described in embodiment 7, wherein the T cell contains an expression frame of the therapeutic protein and an expression frame of the polypeptide that downregulates the cell surface TCR/CD3 complex, or a coding sequence of the therapeutic protein and a coding sequence of the polypeptide that downregulates the cell surface TCR/CD3 complex are in the same expression frame.
9. An engineered T cell, wherein said T cell expresses a polypeptide that downregulates a cell surface TCR/CD3 complex, the polypeptide comprises: an antibody or an antigen binding fragment thereof, or a mutant having at least 90% sequence identity with the antibody or the antigen binding fragment thereof and retaining antigen binding activity of the antibody or the antigen binding fragment thereof; wherein, the antibody comprises a heavy chain variable region and a light chain variable region,
10. The T cell as described in embodiment 9, wherein the polypeptide that downregulates the cell surface TCR/CD3 complex has one or more features selected from the group consisting of:
11. The T cell as described in embodiments 9 or 10, wherein the polypeptide that downregulates the cell surface TCR/CD3 complex has one or more features selected from the group consisting of:
12. The T cell as described in any of embodiments 9-11, wherein,
13. The T cell as described in any of embodiments 9-12, wherein the antibody is a single chain antibody.
14. The T cell as described in any of embodiments 9-12, wherein the polypeptide that downregulates the cell surface TCR/CD3 complex comprises a localization sequence and optionally a signal peptide, preferably,
15. The T cell as described in any of embodiments 9-14, further expresses a therapeutic protein; preferably, the therapeutic protein is a chimeric antigen receptor.
16. The T cell as described in embodiment 15, wherein the T cell contains an expression frame of the therapeutic protein and an expression frame of the polypeptide that downregulates the cell surface TCR/CD3 complex, or a coding sequence of the therapeutic protein and a coding sequence of the polypeptide that downregulates the cell surface TCR/CD3 complex are in the same expression frame.
17. An antibody or an antigen binding fragment thereof, wherein the antibody has an amino acid sequence of HCDR1 of GYKFTSYV, an amino acid sequence of HCDR2 of INX1X2X3DVT, an amino acid sequence of HCDR3 of AX4GX5X6YDYDGFVY, an amino acid sequence of LCDR1 of SSVSY, an amino acid sequence of LCDR2 of DTS, an amino acid sequence of LCDR3 of QQWX7X8X9X10X11T; wherein, X1-X11 are as described in embodiment 10, and HCDR2 is not INPYNDVT, HCDR3 is not ARGSYYDYDGFVY, LCDR3 is not QQWSSNPLT,
18. The antibody or the antigen binding fragment thereof as described in embodiment 17, wherein
19. A fusion protein comprising a localization sequence, a polypeptide that downregulates a cell surface TCR/CD3 complex, and, optionally, a signal peptide; preferably, the polypeptide that downregulates the cell surface TCR/CD3 complex is as described in any of embodiments 9-18, preferably,
20. A nucleic acid molecule, wherein the nucleic acid molecule is selected from the group consisting of:
21. A nucleic acid construct, wherein the nucleic acid construct comprises a nucleic acid molecule as described in embodiment 20,
22. A lentivirus wherein the lentivirus contains a nucleic acid molecule described in embodiment 20 or a nucleic acid construct described in embodiment 21.
23. A host cell, wherein the host cell comprises, expresses and/or secrets an antibody or an antigen binding fragment thereof as described in embodiments 17 or 18, or a fusion protein as described in embodiment 19, and optionally a therapeutic protein; preferably, the host cell comprises a nucleic acid molecule as described in embodiment 20, a nucleic acid construct as described in embodiment 21 and/or a lentivirus as described in embodiment 22.
24. A pharmaceutical composition comprising a T cell as described in any of embodiments 9-16, an antibody or an antigen binding fragment thereof as described in embodiments 17 or 18, a fusion protein as described in embodiment 19, a nucleic acid molecule as described in embodiment 20, a nucleic acid construct as described in embodiment 21 and/or a lentivirus as described in embodiment 22, and a pharmaceutically acceptable carrier.
25. Use of a polypeptide that downregulates a cell surface TCR/CD3 complex or its coding sequence, a nucleic acid molecule as described in embodiment 20, a nucleic acid construct as described in embodiment 21 and/or a lentivirus as described in embodiment 22 in the preparation of a T cell with downregulated cell surface TCR, or in the preparation of T cells for cancer therapy, wherein the polypeptide that downregulates a cell surface TCR/CD3 complex is as described in embodiments 9-14.
The description is further described in detail by reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Thus, the present description should by no means be understood as being limited to the following examples, but rather should be understood as including any and all of the variations that become apparent as a result of the teaching provided herein. The methods and reagents used in the examples, unless otherwise stated, are those conventional in the art.
1. The gene sequences of human CD8α hinge region, human CD8 transmembrane region, 4-1BB intracellular region, human CD3ζ intracellular region, heavy and light chain variable regions of an anti-human CD19 antibody (clone FMC63), and heavy and light chain variable regions of a control anti-human TCR/CD3 antibody (clones UCHT1 and OKT3) were searched from the NCBI website database. The remaining antibody sequences were derived from specific anti-human TCR/CD3 antibody clones. All relevant amino acid sequences were subject to codon optimization at the website https://www.thermofisher.com/order/geneartgenes, which made them more suitable for expression by human cells without a change in the encoded amino acid sequence.
2. Overlapping PCR was used to link above sequences in turn as follows: anti-CD19-scFv gene, human CD8 hinge region gene, human CD8 transmembrane region gene, 4-1BB intracellular region gene, human CD3ζ intracellular region, F2A and CD3-scFv gene, and endoplasmic reticulum retention sequence (AEKDEL) to form complete gene sequence information. Its sequence comprising a coding sequence of a signal peptide was shown as residues 1-66 or 1546-1611 of SEQ ID No: 51, and its amino acid sequence was shown as residues 1-22 and 516-537 of SEQ ID No: 44.
3. The nucleotide sequence of the CAR molecule was seamlessly cloned into the BamHI/EcoRI sites of the lentiviral plasmid pWPXL (Addgene) and transformed into competent E. coli (Stbl3).
4. The recombinant plasmids were sent to Suzhou Genewiz Bio-Technology Co., Ltd. for sequencing, and the sequencing results were aligned with the sequence to be synthesized to verify that the sequence was correct. The sequencing primers were:
After sequencing to be correct, the plasmids were extracted and purified using a plasmid purification kit from Qiagen, and the purified plasmids were transfected into 293T cells by the calcium phosphate method for lentiviral packaging experiments (Molecular Therapy—Methods & Clinical Development, 2016, 3: 16017), the obtained lentiviral vectors were as follows: Control (CAR19-F2A-GFP), Sample 1 (CAR19-F2A-UCHT1 scfv-AEKDEL), Sample 2 (CAR19-F2A-OKT3 scfv-AEKDEL), Samples 3 (CAR19-F2A-CTAB5 scfv-AEKDEL), Sample 4 (CAR19-F2A-CTAB2 scfv-AEKDEL), Sample 5 (CAR19-F2A-CTAB3 scfv-AEKDEL), Sample 6 (CAR19-F2A-CTAB4 scfv-AEKDEL), and Sample 7 (CTAB5 scfv-AEKDEL).
Isolation of PBMCs from peripheral blood, isolation and activation of T cells, lentiviral transduction, ex vivo culture
Healthy donors who tested negative for HBV, HCV, and HIV were selected, blood was drawn from the median cubital vein, the buffer coat of PBMCs was separated by Ficoll density gradient centrifugation, the percentage of CD3+ T cells was determined according to flow cytometry of whole blood, the number of CD3+ T cells was calculated, and a use amount of magnetic beads were taken according to the ratio of Dynabeads CD3/CD28 to CD3+ T cells of 3:1, and incubated with the buffer coat cells for 30 min, CD3+ T cells were isolated, then activated with Dynabeads CD3/CD28 (Life Technology) for 24 hours, and the proportion of CD25+CD69+ T cells was determined by flow cytometry.
After CD3+T activation, lentiviral transduction was performed. The 24 well plates coated with Novonectin were incubated at 37° C. for 2 hours, and the cell suspension was formulated with various lentiviruses prepared as described previously (MOI=3), Tscm (2 U/ml, Novoprotein) into transduction systems, respectively, which were placed in coated 24 well plates, and the cell density was adjusted to 1.0E+06/ml and centrifuged at 500 g for 30 minutes, followed by stationary incubation in an incubator at 37° C. and 5% CO2 for 48 hours. After lentiviral transfection, the cells were cultured in Xvivo15 medium with 5% FBS, supplemented with Tscm (to final concentration of 2 U/ml) every other day, counted, and adjusted to 0.5E+06/ml, and harvested after culture until Day 8-10.
5.0E+05 cells were counted and collected, and the cells were washed 2 times and resuspended in 100 ul of buffer containing 4% BSA. 8 ul of anti-human TCR or CD3 antibody was added to each tube of cells, vortexed to mix, and incubated at 4° C. for 30 minutes. After staining and incubation, the cells were washed repeatedly, and fluorescently labeled anti-CAR19 antibody or Protein L was diluted 500×, and cells were resuspended in 200 ul per tube by vortexing and incubated at 4° C. for 30 minutes. The cells were washed repeatedly and resuspended in 500 ul of buffer containing 4% BSA, 4 ul of 7AAD dye was added to each tube, vortexed to mix, and incubated at room temperature for 10 minutes in dark. Finally, the samples were transferred to flow tubes, and the efficiency of CAR19 transfection as well as the surface TCR or CD3 levels of CAR19+ T cell populations were detected on Calibr flow cytometer.
Two widely used murine derived anti-human TCR/CD3 antibodies (OKT3 and UCTH1) were first evaluated. To avoid rejection by murine derived sequences in clinical applications, they were humanized and tested at the cellular level. The results were shown in
The activity on downregulating the cell surface TCR/CD3 complex of the polypeptide containing the humanized OKT3 sequence is higher than that of the polypeptide containing the UCH1 sequence (
Unexpectedly, a polypeptide containing the CTAB5 sequence of the antibody was able to most efficiently downregulate the level of the cell surface TCR/CD3 complex, with up to 88.09% of CAR+ cells expressing no surface TCR; up to 85.89% of CAR+ cells expressing no surface CD3 (
athe proportion of Q4/the proportion of Q2 + Q4
The CAR-T cells from different donors containing CTAB5 sequence polypeptides were prepared according to the procedure of Examples 3 and 4, and the efficiency of CAR19 transfection as well as the TCR or CD3 mean fluorescence intensity of the CAR19+ T cell populations were determined by flow cytometry according to Example 5.
Results were as shown in
athe proportion of Q4/the proportion of Q2 + Q4
To confirm the activity of the polypeptide containing CTAB5 antibody sequence, lentiviral vectors expressing only the polypeptide containing CTAB5 sequence but not the therapeutic protein were prepared as in Example 2, the corresponding T cells were prepared as in Examples 3 and 4, and the TCR or CD3 levels on their surfaces were detected by flow cytometry as in Example 5.
Results were as shown in
The CAR-T cells in each group were counted, and the 1.0E+06 cells were collected by centrifugation, and the cells were washed twice by resuspension with sterile 4% BSA, followed by resuspension with 200 ul 4% BSA. Anti-human antibodies as follows were added to each sample: CD4 (AmCyan), CD8 (APC-Cy7), CD45RA (PE-Cy7) and CCR7 (BV421). 5 ul of each antibody was added, mixed well by vortex homogenizer and incubated at 4° C. for 30 minutes. After staining and incubation, the cells were washed repeatedly and resuspended with 500 ul of 4% BSA buffer, 4 ul of 7AAD dye was added to each tube, vortexed and incubated at room temperature in dark for 10 minutes. Samples were transferred to flow tubes, and T cell immunophenotypes were analyzed for CCR7 and CD45RA expression on T cells from each group on FACS Calibr flow cytometer.
The results were shown in
Each group of T cells transfected with lentiviral vector, T cells were counted every other day, T cells were cultured in vitro to Day 8, and the cell proliferation fold (cell number on Day 8/cell number used for transfection) was calculated.
The results were shown in
NC-T cells (T cells without lentiviral transfection) and CAR-T cells in each group were counted and collected, and the cell density was adjusted to 5.0E+07/ml with T cell medium X-VIVO15 (without Tscm). The target cells were counted and collected, and the cell density was adjusted to 5.0E+06/ml with RPMI 1640 (without FBS). The above adjusted densities of effector cells were mixed with target cells at different effector to target ratios (1:1, 2.5:1, 5:1, 10:1, 20:1) in a 1.5 ml centrifuge tube, and the total volume was made up to 200 ul with X-VIVO15. They were moved into 96 well V-bottom plate and incubated at 37° C. and 5% C02 for 24 h, gently pipetted to mix the cells of each well and 100 ul of cell suspension was transferred into a 96 well plate with white wall solid bottom. 80 μl ONE-Glo™ Luciferase substrate was added, mixed by pipetting, and detected for the fluorescence intensity on Luminoskan Ascent chemiluminescence analyzer after incubation in dark at room temperature for 10 minutes.
The results were shown in Table 3 and
Lentivirus transfected T cells from each group were cultured in vitro until Day 8, and the TCR dependent T cell activating antibody OKT3 was added to final concentrations as follows: 50 ng/ml, 100 ng/ml, 150 ng/ml, and 200 ng/ml. Each group of CAR-T cell samples was diluted by appropriate fold with X-VIVO15 media, corresponding number of strips were removed as required, 100 ul of dilution RD1-51 was added to each well, diluted samples were mixed and 100 ul was taken into wells (completed within 15 minutes); covered with a parafilm and incubated for 2 hours at room temperature. After the incubation was complete, the solution in the wells was completely removed, wash solution was added, standed for 1 minute, this step was repeated for a total of four washes; 200 ul of human IFN-γ coupling reagent was added to each well, covered with a new parafilm and incubated for 2 hours at room temperature; 200 ul of substrate solution was added to each well and incubated for 30 minutes in dark at room temperature; after the incubation was completed, 50 ul of stop solution was added to each well, mixed well and then detected on a microplate reader (wavelength set at 450 nm, corrected at 570 nm/540 nm).
The results were shown in Table 4 and
1) The target TCR/CD3 antibody sequence was selected for amino acid point mutations to search for mutants that more effectively downregulated surface TCR/CD3 complex expression, or eliminated its MHC-I epitope, or could promote application advantages such as T cell expansion. Region 1 of the mutant library was located in the light chain of the target scFv (amino acids 90-95 of SEQ ID No: 43), region 2 was located in the heavy chain of the target scFv (amino acids 50-55 of SEQ ID No: 42), and region 3 was located in the heavy chain of the target scFv (amino acids 97-101 of SEQ ID No: 42).
2) NNK degenerate primers were designed to carry out the construction of above mutation library. The resulting library of lentiviral target mutants was prepared following the procedure of Examples 1 and 2.
3) The resulting mutant T cell library of interest was prepared following the procedure of Examples 3 and 4.
4) The TCR and CD3 expression profile of the target mutant T cell library was examined as in Example 5. Results were as shown in
5) The subset of T cell clones with the highest capacity to downregulate TCRs was flow sorted, and genomic high-throughput sequencing was performed. Table 5 shows 23 exemplary mutants in the library. Three mutants of them were selected and confirmed to be able to down-regulate the expression of the cell surface TCR/CD3 complex, and effectively kill target cells.
6) The selected mutants: mutant 1, VL-CDR3 (SEQ ID No: 23); mutant 2, VL-CDR3 (SEQ ID No: 24); mutant 3, VH-CDR3 (SEQ ID No: 17). Corresponding point mutation vectors were constructed.
7) The relevant CAR-T indicators of the 3 mutants were detected following the procedure of above examples, and the results were shown in
According to
All documents mentioned in the present description are cited in the present application as references, as if each is cited separately as a reference. It is further understood that, after reading the above contents of the present description, those skilled in the art may make various changes or modifications to the present description, and these equivalent forms still fall within the scope defined by the claims appended to the present application.
Sequence in the Description
| Number | Date | Country | Kind |
|---|---|---|---|
| 202010022475.6 | Jan 2020 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/070820 | 1/8/2021 | WO |
