Patent Application
20200306303
The present invention relates to adoptive cell therapy using Akt-overexpressing immune cells. More specifically, the Akt-overexpressing immune cells can be utilized for treatment of viral infection and malignancies in immunosuppressive microenvironment.
Adoptive cell therapy (ACT) utilizing gene engineering to introduce antigen specificity or to enhance effector functions or survival of immune cells is feasible and high clinical values for treatment of chronic infections or malignancies since virus- or tumor-specific immune cell response is usually impaired or missing in patients with most of these chronic diseases.
However, during chronic viral infections or malignancies, there are usually monoclonal T cell response detected and most of the antigen-specific T cells undergo exhaustion or apoptosis rapidly after activation. It is often observed that the virus or tumor-specific cytotoxicity T lymphocytes (CTLs) undergo T-cell exhaustion due to persistent T-cell receptor (TCR) signaling and lack of suitable co-stimulation. T cell exhaustion features the gradual loss of proliferative capability and cytokine production, impaired cytotoxicity, surface expression of various immune checkpoints and increase of apoptotic rate[1, 2].
Immune checkpoints e.g. PD-1 and CTLA-4 are molecules up-regulated on T cells in response to TCR signaling to modulate the extent of T-cell activation and are highly expressed on exhausted T cells. It has been shown in several studies that signaling through immune checkpoints on T cells can impair metabolic reprogramming during T-cell activation and differentiation[3-6].
The molecular pathways by which most of the immune checkpoints signal remain poorly understood except that PP2A and SHP2 activated by PD-1 and CTLA-4 signaling, respectively, can suppress Akt activation of T cells upon TCR stimulation, being revealed[7].
Akt is shown to have a great influence on T-cell growth, proliferation, and survival and also demonstrated to be a signal integrator for T-cell differentiation through regulation of Foxo, mTOR and Wnt/β-catenin pathways[8-11]. During chronic LCMV infection, the activation of Akt and mTOR signaling in CTLs is impaired, which results in T-cell exhaustion through PD-1 signaling in virus-specific CTLs[12].
Therefore, the present invention demonstrates that reinforcement of Akt/mTOR pathway in anti-viral or anti-tumor CTLs may rescue them from T cell exhaustion and has the potential to be further applied on recombinant TCR technology or chimeric antigen receptor (CAR) technology [13] to enhance the survival and effector functions of engineered T cells for treatment of patients with malignancy or chronic viral infection.
The present invention provides a method able to enhance survival and functionality of anti-tumor or anti-viral T cells through overexpression of Akt molecules in CTLs. The Akt-overexpressing CTLs are shown to have high proliferative capability and superior effector functions during encounter with the antigen in the liver, which suggests that the Akt molecules can help the CTLs to overcome T-cell exhaustion in the inhibitory microenvironment. We further show expression of Akt molecules can facilitate anti-viral and anti-tumor CTL responses e.g. proliferation, cytokine production and cytotoxicity. Moreover, it enables the CTLs resistance to proliferative arrest induced by MDSCs. the expression of constitutively active Akt molecules enable T cells to gain the privilege to survive and to kill in the tolerogenic liver or tumor microenvironments. The active Akt molecules only when in combination with TCR signaling can trigger massive proliferative response of CTLs and therefore are safe to be applied to T-cell engineering of CTLs.
In one embodiment, this present invention demonstrates that the myristorylated Akt molecules are able to anchor on cell membrane and can be phosphorylated. After being adoptive transfer into the recipient mice, Akt1- and Akt2-CTL populations expand vigorously in the liver and the spleen. It indicates overexpression of Akt is related to intrahepatic survival or secondary expansion of CTLs in response to antigen stimulation.
T cell exhaustion features surface expression of various immune checkpoints. The immune checkpoint blockade can rescue T cell exhaustion of CTLs and further enhance the anti-tumor responses. In another embodiment, this present invention demonstrated that Akt signaling prevents the expression of immune checkpoints, especially LAG-3 and TIGIT on HBV-specific CTLs.
In some embodiments, this present invention demonstrates that Akt1/2-engineered CTLs clear intrahepatic viral infections efficiently in two different models and persist and provide protective memory immunity in the recovered individuals.
In some embodiments, Akt2-engineered CTLs are able to eradicate established liver cancers in an oncogene-induced HCC mouse model. AKT1 and AKT2 genes can be utilized in T-cell engineering of adoptive T-cell therapy for treatment of hepatic chronic viral infection and malignancies since Akt signaling is able to reverse T-cell exhaustion of CTLs in immunosuppressive microenvironment.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention belongs.
As used herein, the term “OT-I cell” refers to a transgenic line of ovalbumin-specific, CD8+ T cell. The transgenic T cell receptor was designed to recognize ovalbumin residues 257-264 in the context of H-2Kb and used to study the role of peptides in positive selection and the response of CD8+ T cells to antigen.
As used herein, the term “AdHBV” refers to the adenovirus carrying HBV genome. HBV-infected mouse model can be established by hydrodynamic injection (HDI) of the HBV genome into the tail vein.
As used herein, the term “HBcAg” refers to a hepatitis B viral protein, which is an antigen that can be found on the surface of the nucleocapsid core of the hepatitis B virus.
As used herein, the term “HBeAg” refers to a hepatitis B viral protein, which is an antigen that can be detected in the serum of mice with HBV infection established by AdHBV infection or HDI of a plasmid harboring the HBV genome.
The DNA or RNA molecules in this present invention can be amplified through plasmid amplification, in vitro transcription or in vitro synthesis and transfected into target cells through electroporation, liposome or other chemical vehicles.
The aforementioned target cells for genetic modification can be T cells, nature killer cells, hematopoietic stem cells, embryonic stem cells and pluripotent stem cells from various species. These cells can be modified by viral transduction or DNA (or RNA) transfection.
The recombinant viral or transposon vectors can be retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, other related viruses and various transposon systems can be used in transduction or integration of transgenes.
To investigate the mechanism of how liver microenvironment can influence secondary expansion of virus-specific CTL population in the liver, in vitro-activated HBV specific CD8+ T cells are adoptively transferred into HBV carrier mice and the change of the serum level of HBV antigen in these mice is detected. It is found that most of the mice failed to eliminate persistent HBV infection within 42 days. The cell number and expression level of exhaustion markers including PD-1, TIM-3, and LAG-3 on the adoptively transferred CTLs in the liver and in the spleen of the HBV carrier mice are further detected. The cell number of adoptively transferred HBV-specific CTLs increases in the liver but not in the spleen. The HBV-specific CTLs in both the liver and the spleen express higher levels of PD-1 and LAG-3 than endogenous CD8+ T cells; however, the splenic HBV-specific CTLs express lower levels of PD-1. TIM-3 and LAG-3 than intrahepatic compartments. Those results demonstrate that the exposure to HBV antigens expressed in the liver microenvironment induces T-cell exhaustion of HBV-specific CTLs.
The immune checkpoints PD-1 and CTLA-4 are shown to prevent Akt phosphorylation % activation during TCR triggering through recruitment of SHP-1/2 and activation of PP2A, respectively. We therefore examine whether Akt signaling is critical to intrahepatic expansion and differentiation of CD8+ T cells. Mouse AKT1, AKT2 and AKT3 genes are cloned, respectively, with addition of src myristoylation sequence in the upstream of AKT genes to ensure the membrane targeting and being constitutively active of the Akt molecules. The expression of exogenous myristoylated Akt isoforms are detected by Western blot in Akt-engineered CTLs but not in the control T cells. CTLs are engrafted with three different kinds of Akt, respectively, all show Akt phosphorylation at Ser473 and only those are engrafted with Akt1 or Akt2 show Akt phosphorylation at Thr308.
To examine whether overexpression of Akt is related to intrahepatic survival or secondary expansion of CTLs in response to antigen stimulation, the ovalbumin (OVA) and luciferase expression are induced in the liver of recipient mice by hydrodynamic injection (HDI) of a plasmid encoding OVA and luciferase. After being adoptive transfer into the recipient mice, Akt1- and Akt2-engineered CTL populations expand vigorously in the liver and the spleen. There is more than 250,000-fold for Akt1 CTLs and 950,000-fold for Akt2-CTLs cell numbers found in the liver in comparison with that of ctrl-CTLs at day 7 after adoptive transfer.
Owing to the huge contribution of immune checkpoints on T-cell exhaustion in the liver during chronic viral infection, the inventors therefore examine whether Akt signaling have an influence the expression of immune checkpoint molecules on HBV-specific CTLs per se. After in-vitro activation and transduction, the Akt- or ctrl-engineered HBc93-100-specific CTLs are adoptively transferred into AdHBV-infected mice and analyzed the surface expression of immune checkpoint molecules on the CTLs at day 6 and day 19 after adoptive transfer. Hepatic ctrl-CTLs expressed high level of PD-1, TIM-3 and LAG-3 at day 19 after adoptive transfer, whereas Akt1-CTLs and Akt2-CTLs expressed significantly less PD-1, TIM-3 and LAG-3 at day 19 post adoptive transfer.
To further investigate whether these Akt-CTLs can overcome the suppressive mechanisms in the liver and mediate clearance of persistent HBV infection, the ctrl- or Akt1-engineered HBc93-100-specific CTLs are adoptively transferred into HBV carrier mice. Akt1-CTLs but not ctrl-CTLs eliminate persistent HBV infection within 14 days after being adoptive transferred into HBV carrier mice. The Akt1-CTLs are mainly in the liver rather than in the spleen and disperse to the spleen after antigen clearance. There are less HBcAg-positive hepatocytes but more cleaved caspase 3-positive apoptotic hepatocytes detected in the liver of mice receiving Akt1-CTLs than in the liver of mice receiving ctrl-CTLs. After clearance of antigen, the mononuclear cells reduce and HBcAg-positive hepatocytes as well as cleaved caspase 3-positive hepatocytes are no longer detected in the liver of mice receiving Akt1-CTLs. The ctrl-CTLs fail to clear HBV and do not induce significant inflammation after being adoptively transferred into HBV carrier mice. Akt2-CTLs expand vigorously when encountering the cognate antigen in vivo, and prevent T-cell from exhaustion. Also, Akt2-CTLs exhibit strong cytotoxic function and are more efficient to clear HBV infection than ctrl CTLs.
The capability of Akt-engineered CTLs in killing of hepatocellular carcinoma (HCC) is further examined. The tumor antigen-specific Akt2-engrafied CD8+ CTLs can accumulate in the tumor sites as well as in the liver at day 10 after adoptive transfer into HCC-bearing mice. These Akt2-CTLs change the tumor microenvironment and to attract or activate the surrounding F4/80+ macrophages in tumor sites. Furthermore, a lot of cleaved caspase 3-positive tumor cells are detected in the mice receiving Akt2-CTLs but not in ctrl mice. Elevated serum ALT in the mice with Akt2-CTLs is also observed but not in ctrl mice (118.1 U/L vs. 22.8 U/L). It can be concluded that Akt2 activation enables CTLs to have strong effector functions and be able to kill tumor cells in the liver. This is probably through CTLs' own cytotoxic capability or through release of cytokines to activate the anti-tumor functions of tumor-associated macrophages.
To further explore the potential application of Akt molecules on cancer immunotherapy, the plasmids carrying human or mouse Akt1 or Akt2 genes and anti-CEA (Carcinoembryonic antigen) chimeric antigen receptor (CAR) are constructed. CEA are glycosyl phosphatidyl inositol (GPI) cell-surface-anchored glycoproteins and are critical to the dissemination of colon carcinoma cells. The modified CTLs are co-cultured with a colorectal adenocarcinoma cell line, LS174T. Both CD4+ and CD8+ T cells with the engraftment of anti-CEA CAR can respond to stimulation of LS174T and proliferate. Additional active Akt1 expression in anti-CEA CAR engrafted T cells can promote the proliferation capability of both CD4+ and CD8+ T cells. More IL-2 and IFNγ are detected in the culture medium of co-culture of LS174T cell line with T cells expressing anti-CEA CAR and Akt1 or Akt2 molecules compared to that of LS174T and T cells expressing solely anti-CEA CAR. Intracellular staining of IFNγ and granzyme B of the CD8+ T cells co-culture with LS174T cells also proves that Akt1 or Akt2 overexpression can enhance the cytokine production and cytotoxicity in CTLs. Strikingly, Akt1- and Akt2-overexpressing CTLs, respectively are shown to have the capability to overcome the proliferative arrest induced by myeloid-derived suppressor cells (MDSCs), which strongly suggests that the potential application of Akt molecules on T-cell engineering technology e.g. CAR T cells for immunotherapy.
The following examples are offered by way of illustration and not by way of limitation. The mAkt isoforms are utilized in the mouse model as a demonstration in this present invention, but is not intended to limit the scope of the invention.
In vitro-activated CD45.1 HBc93-100 specific CD8+ T cells are adoptively transferred into congenic C57BL/6 mice infected with the adenovirus carrying HBV genome (AdHBV), and the change of the serum level of HBeAg in these mice is detected. It is found that most of the mice failed to eliminate persistent HBV infection within 42 days (
The cell number and expression level of exhaustion markers are further detected, which including PD-1, TIM-3, and LAG-3 on the adoptively transferred CTLs in the liver and in the spleen of the HBV carrier mice at day 3, day 7 and day 14 post adoptive transfer. The cell number of adoptively transferred HBV-specific CTLs increases from day 3 to day 14 in the liver but not in the spleen (
Endogenous CD8+ T cells are used as a reference population for evaluation of the expression level of these exhaustion markers on HBV-specific CTLs. The HBV-specific CTLs in both the liver and the spleen express higher levels of PD-1 and LAG-3 than endogenous CD8+ T cells but no or little TIM-3 at day 3 and day 7 post adoptive transfer (
The splenic HBV-specific CTLs express lower levels of PD-1 and LAG-3 than intrahepatic compartments at all time points (
Murine stem cell retroviral (MSCV) system is chosen for delivery of genes into T lymphocytes due to its high efficiency to transduce hematopoietic cell lineages. A pMSCV-CD90.1 plasmid is generated from a replacement of hygromycin resistance gene by p2A peptide sequence and mouse CD90.1 open reading frame (ORF) with the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) in the 3′ untranslated region of CD90.1 gene to enhance the expression of the transgenes. The CD90.1 gene and WPRE sequence are amplified from pLKO_TRC024 plasmid (RNAi core lab, Taipei, Taiwan). Mouse AKT1 (SEQ ID NO: 1). AKT2 (SEQ ID NO: 3) and AKT3 (SEQ ID NO: 5) genes are cloned, respectively, through PCR using cDNA from mouse 4T1 breast cancer cells with addition of src myristoylation sequence by PCR primer in the upstream of AKT genes to ensure the membrane targeting and being constitutively active of the Akt molecules. The myristoylation sequence and AKT genes are linked, respectively, to mouse CD90.1 gene by p2A peptide sequence in pMSCV-CD90.1 to result in pMSCV-mAkt1-CD90.1, pMSCV-mAkt2-CD90.1 and pMSCV-mAkt3-CD90.1. The expression cassette is flanked by 5′ and 3′ MSCV long terminal repeats (LTRs). The 4 plasmids are used to produce recombinant retroviruses carrying mouse AKT1. AKT2, AKT3 or control CD90.1 gene, respectively (
Splenic ovalbumin-specific TCR tg OT-I CD8+ T cells are activated by anti-CD3+anti-CD28 beads, subsequently transduced by recombinant retroviruses and are subjected to surface marker staining using antibody recognizing CD90.1 as a tag for transgene expression followed by flow cytometric analysis. Around 75% to 95% of the effector CD8+ T cells are transduced with retroviruses carrying CD90.1, AKT1-CD90.1 or AKT2-CD90.1 gene, positive for CD90.1, whereas only 23% of the cells are transduced with retroviruses carrying AKT3-CD90.1 gene expressed low level of CD90.1 (
It has been shown that the expression patterns of the three Akt isoforms are different. Akt1 (SEQ ID NO: 1) and Akt2 (SEQ ID NO: 3) are ubiquitously expressed in nearly all tissues whereas Akt3 (SEQ ID NO: 5) are mainly expressed in brain and testes. The tissue specific expression manner of Akt isoforms may explain the low expression of Akt3 by the CD8+ T cells. The expression of exogenous myristoylated Akt isoforms is detected by Western blot in Akt-engineered CTLs but not in the control T cells. CTLs engrafted with three different kinds of Akt, respectively, all show Akt phosphorylation at Ser473 and only those which are engrafted with Akt1 or Akt2 show Akt phosphorylation at Thr308 (
Ovalbumin (OVA) and luciferase expression are induced in the liver of recipient mice by hydrodynamic injection (HDI) of a plasmid encoding OVA and luciferase under the control of albumin promoter (pENTRY-Albp-OL). After being adoptive transfer into the recipient mice, Akt1- and Akt2- but not Akt3-engineered CTL or CD90.1-engineered (ctrl) populations expanded vigorously in the liver and the spleen.
These Akt1- or Akt2-CTLs underwent vigorous proliferation and yielded 23 million (Akt1) and 113 million (Akt2) splenic and intrahepatic CTLs in total, respectively, after antigen stimulation in the liver (
The massive expansion of Akt1- or Akt-2-OT-I CTLs is further confirmed in a time kinetic experiment (
Therefore, Akt constructs with co-expression of luciferase instead of CD90.1 are designed for monitoring the distribution and expansion of Akt-engineered CTLs. Control (ctrl) Luc-CTLs and Akt2-Luc-CTLs are delivered respectively, to mice with or without OVA expression in their livers and only observed TCR signaling-dependent Akt2-Luc-CTL accumulation in the liver but not in other organs or in mice without antigen expression in the liver (
After in-vitro activation and transduction, HBc93-100-specific CD8+ T cells at day 3 after activation are analyzed for their surface expression of various immune checkpoints. The overexpression of constitutively active Akt12 does not change the surface expression of PD-1 and TIGIT (
These CTLs at day 3 after anti-CD3/anti-CD28 bead activation may have returned to resting status with low or no expression of immune checkpoints e.g. PD-1 and TIGIT except LAG-3. Therefore, the expression level of these immune checkpoints on CTLs after re-stimulation is measured. Expression of PD-1 is rapidly detected on ctrl-, Akt1- and Akt2-CTLs (
To further investigate whether the regulation of immune checkpoints on CTLs by Akt signaling also happens in liver microenvironment, the Akt1- or ctrl-engineered HBc93-100-specific CTLs are adoptively transferred into AdHBV-infected mice and analyzed the surface expression of immune checkpoint molecules on the CTLs at day 6 and day 19 after adoptive transfer. The expression patterns of each examined immune checkpoints are quite different. Both intrahepatic Akt1- and ctrl-engineered CTLs at day 6 after adoptive transfer express high level of PD-1 when encountering the cognate antigen in the liver, but the PD-1 expression is down regulated in the Akt1-CTLs at day 19 after adoptive transfer (
At day 6 after exposure to HBV, a certain proportion of the hepatic Akt1-CTLs expressed high level of TIM-3, whereas splenic CTLs and ctrl-CTLs in liver express lower level of TIM-3 at this time point, which suggests a stronger TCR triggering in Akt1-CTLs than in ctrl-CTLs (
Hepatic ctrl-CTLs express high level of LAG-3 at both day 6 and day 19 after adoptive transfer, whereas Akt1-CTLs express less LAG-3 on their surface during the whole period (
These in-vitro and in-vive data clearly demonstrate that Akt signaling possesses very few influence on PD-1 expression but positively regulates TIM-3 expression on CTLs during early TCR signaling. We further prove that augmentation of Akt signaling prevents the expression of LAG-3 and TIGIT on CTLs in the liver during persistent HBV infection, which may contribute the robust expansion and potent effector functions of Akt-CTLs against HBV.
The higher expression of PD-1 and TIM-3 on Akt-CTLs than on ctrl-CTLs after re-stimulation in vitro and in vivo strongly suggests a stronger TCR triggering in Akt-CTLs than that in ctrl-CTLs and also excludes the lack of antigen stimulation at this early time point, which results in down-regulation of LAG-3 and TIGIT. The early expression of TIM-3 on Akt-CTLs may additionally involve in the augmentation of effector functions of Akt-CTLs to combat HBV infection. The reduced expression of immune checkpoints on Akt-engineered CTLs at the later time point may result from the lack of antigen stimulation due to the intense effector functions of Akt-CTLs, which facilitates the early removal of the HBV antigen from the liver.
The cell number of adoptively transferred ctrl- or Akt1-engineered HBc93-100-specific CTLs in the liver and in the spleen of HBV carrier mice is measured, and there are more Akt1-CTLs than ctrl-CTLs recovered from the liver at both of day 6 and day 19 after adoptive transfer (
Akt1-CTLs but not ctrl-CTLs eliminate persistent HBV infection within 14 days after being adoptive transferred into HBV carrier mice (
Immunohistochemical staining is performed to visualize the HBcAg or cleaved caspase 3 expression by hepatocytes and immune cells in the liver of HBV carrier mice. There are less HBcAg-positive hepatocytes but more cleaved caspase 3-positive apoptotic hepatocytes detected in the liver of mice receiving Akt1-CTLs than in the liver of mice receiving ctrl-CTLs at day 6 after adoptive transfer (
After clearance of antigen, the liver histology appears back to normal, the mononuclear cells reduce and HBcAg-positive hepatocytes as well as cleaved caspase 3-positive hepatocytes are no longer detected in the liver of mice receiving Akt1-CTLs (
Akt2-CTLs also expand vigorously when encountering the cognate antigen in vive (
We further examined the capability of the engineered CTLs to eliminate antigen from the liver through the measurement of the bioluminescence in the liver of the recipient mice. The loss of bioluminescence represented the clearance of antigen from the liver. We found that Akt1-OT-I CTLs were more efficient than ctrl OT-I CTLs to eliminate OVA from the liver (
Being concerned about that the overexpression of Akt molecules in CTLs may potentially induced oncogenic property of the transduced cells, we therefore monitored the numbers of intrahepatic and splenic transferred CTLs and serum ALT levels in the mice receiving ctrl-CTLs and Akt1-CTLs for a longer period of time. The serum ALT levels of mice receiving Akt1-CTLs decreased to normal levels after the clearance of antigens and cell numbers of Akt1-CTL also dropped at least 5000-fold from day 7 to day 63 (
We further analyzed the proliferation capability of these adoptively transferred Akt1-CTLs or endogenous CD8+ T cells at day 7 and day 63, respectively and found that even in the absence of antigen, the Akt1-CTLs could still undergo higher grade DNA synthesis to sustain self-renewal than endogenous CD8+ T cells did, which explained the maintenance of the cell number after clearance of antigen (FIGS. 10H and 10I). These Akt1-CTLs in the liver sinusoid were all Ki-67-positive at day 7 after adoptive transfer, which demonstrated that they were undergoing vigorous proliferation and were barely detected in the liver sinusoid at day 32 and day 63 after adoptive transfer (
It has been shown that virus-infected hepatocytes were highly sensitive to CTL-induced cytotoxicity. The liver microenvironment after HDI may not completely mimic that during viral infection. We therefore established an adenovirus (Ad-Albp-OL)-based liver infection mouse model with persistent expression of OVA and luciferase only in the liver under the transcriptional control of albumin promoter in order to study the functions of Akt in CTLs under the circumstance of intrahepatic persistent viral infection. We first titrated the viral doses for infection and found that infection with 2×108 and 4×108 iu of Ad-Albp-OL, respectively, could induce stable expression of luciferase for more than 2 months (
Similar to the data from HDI model, there were more Akt1- or Akt2-CTLs than ctrl-CTLs detected in the liver and in the spleen of Ad-Albp-OL-infected mice at day 7 after adoptive transfer (
Similar result was observed in the mice receiving Akt2-CTLs (
The capability of Akt-engineered CTLs in killing of hepatocellular carcinoma (HCC) is further examined and demonstrated that the tumor antigen-specific Akt2-engrafted CD8+ CTLs can accumulate in the tumor sites as well as in the liver at day 10 after adoptive transfer into HCC-bearing mice (
A lot of cleaved caspase 3-positive tumor cells are detected in the mice receiving Akt2-CTLs but not in ctrl mice (
Ctrl-, Akt1- and Akt2-engineered HBc93-100-specific CTLs are adoptively transferred into HCC-bearing mice, respectively. The oncogenes-induced HCC mouse model is engineered to express luciferase and surrogate tumor antigen-HBc93-100 peptide in the tumor. The tumor growth can be monitored using IVIS and demonstrate that Akt2- but not ctrl- or Akt1-CTLs effectively eliminate HCC as shown by the reduction of in vivo bioluminescence and the disappearance of tumor nodules in the livers of mice receiving Akt2-CTLs (
It can be concluded that Akt2 activation enables CTLs to have strong effector functions to kill tumor cells in the liver.
To further explore the potential application of Akt molecules on cancer immunotherapy, plasmids carrying human or mouse Akt1 or Akt2 genes are constructed and the ORF encoding anti-CEA chimeric antigen receptor (CAR) (
The modified CTLs are co-cultured with a colorectal adenocarcinoma cell line with the expression of CEA, LS174T and the proliferation of the CTLs is monitored through detection of incorporation of a thymidine analog, EdU. Both CD4+ and CD8+ T cells with the engraftment of anti-CEA CAR can respond to stimulation of LS174T and proliferate. Akt signaling further enhances the proliferative capability of anti-CEA CAR-engrafted CD4+ and CD8+ T cells (
Higher levels of IL-2 and IFNγ are detected in the culture medium of co-culture of LS174T cell line with T cells expressing anti-CEA CAR and Akt1 or Akt2 molecules compared that of T cells expressing solely anti-CEA CAR (
Akt1-overexpressing and Akt2-overexpressing CTLs are shown to have the capability to overcome the proliferative arrest induced by myeloid-derived suppressor cells (MDSCs) (
This present invention provides a method able to enhance survival and functionality of anti-tumor or anti-viral T cells through overexpression of Akt molecules in CTLs. The Akt-overexpressing CTLs are shown to have high proliferation capability and superior effector functions during encounter with the antigen in the liver, which suggests that the Akt molecules can help the CTLs to overcome T-cell exhaustion in the inhibitory microenvironment. This present invention further shows expression of Akt molecules can facilitate anti-viral and anti-tumor CTL responses e.g. proliferation, cytokine production and cytotoxicity. Moreover, it enables the CTLs resistance to proliferative arrest induced by MDSCs. To sum up, the expression of constitutively active Akt molecules enable T cells to gain the privilege to survive and to kill in the tolerogenic liver or tumor microenvironments. The active Akt molecules only when in combination with TCR signaling can trigger massive proliferative response of CTLs and therefore are safe to be applied to T-cell engineering of CTLs. Inventors therefore have the following claims for the compositions comprising the anti-tumor or anti-viral engineered T cells and the methods using thereof for treatment of chronic viral infections and malignancies.
This application claims priority to U.S. Provisional Application No. 62/565,820, filed on SEP 29, 2017, the entire content of which is hereby incorporated by reference herein in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2018/053692 | 10/1/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62565820 | Sep 2017 | US |
