Patent Application
20240325531
The present invention refers to the medical field. Particularly, the present invention refers to CAR (chimeric antigen receptor) T/NK-cells for use in the treatment of fungal infections.
Fungi are ubiquitous environmental organisms with special clinical relevance in immunocompromised individuals and hospitalized patients. The spectrum of fungal diseases in humans ranges from mild skin or mucosal infections to life-threatening invasive fungal infections (IFIs). Mortality and morbidity rates due to fungal infections are still rising as a consequence of multiple factors, comprising: (a) the increasing number of surgical procedures and subsequent augment of intensive care units (ICU) admitted patients, (b) the emergence of multidrug resistant (MDR) pathogens, (c) clinical interventions triggering neutropenia, (d) the AIDS epidemics, and (e) population aging.
Current antifungal treatment effectiveness depends on drug selection and diagnosis. In this context, azoles, echinocandins and polyenes are the main antifungal agents used for IFIs treatment. More specifically, ergosterol and 1,3-β-glucan biosynthesis inhibitors, such as fluconazole and caspofungin, respectively, are used as first-line therapeutic agents.
However, these compounds have multiple side effects including cross-resistance, toxicity and drug interactions.
So, there is an unmet medical need of finding alternative antifungal therapies, which can be used to efficiently treat fungal infection without causing relevant side effects.
The present invention is focused on solving this problem and a new antifungal therapy is herein in proposed, which is based on the adoptive cell transfer of autologous Tαβ or allogeneic Tγδ or NK cells expressing a specific CAR.
As explained above, the present invention refers to autologous/allogeneic CAR T/NK-cells, comprising a specific CAR, for use in the treatment of fungal infections.
Particularly, the inventors of the present invention have developed specific CAR T/NK-cells for adoptive cell immunotherapy to severe fungal infections. The autologous/allogeneic CAR T/NK cells of the present invention have been made by transducing these cells with a lentiviral vector comprising a nucleic acid encoding SEQ ID NO: 1.
Such as it can be seen in the Examples provided below, these CAR T/NK cells have been tested in vitro and in vivo, showing an improved survival of mice infected with Candida albicans. So, the effect of the CAR T/NK over C. albicans is herein used as proof of concept, but this therapy could be efficiently used against other invasive fungal infections, preferably caused by fungal species from the genus Candida, Aspergillus, Fusarium, Cryptococcus,
So, the first embodiment of the present invention refers to a CAR (hereinafter CAR of the invention) comprising an extracellular domain which (in turn) comprises SEQ ID NO: 1. In other words, the extracellular domain of the CAR of the invention is characterized by comprising SEQ ID NO: 1.
In a preferred embodiment, the CAR of the invention comprises an extracellular domain which consists of the SEQ ID NO: 1 or SEQ ID NO: 2. So, in a preferred embodiment, the extracellular domain of the CAR of the invention is characterized by consisting of SEQ ID NO: 1 or SEQ ID NO: 2.
VYLKDGWHMVCSQSWGRSSKQWEDPSQASKVCQRLNCGVPLSLGPFLVT
YTPQSSIICYGQLGSFSNCSHSRNDMCHSLGLTCLEPQKTTPPTTRPPP
TTTPEPTAPPRLQLVAQSGGQHCAGVVEFYSGSLGGTISYEAQDKTQDL
ENFLCNNLQCGSFLKHLPETEAGRAQDP/LGEPREHQPLPIQWKIQNSS
CTSLEHCFRKIKPQKSGRVLALLCSGFQPKVQSRLVGGSSICEGTVEVR
QGAQWAALCDSSSARSSLRWEEVCREQQCGSVNSYRVLDAGDPTSRGLF
CPHQKLSQCHELWERNSYCKKVFVTCQDPNP
In a preferred embodiment, the CAR of the invention further comprises a signal peptide, a transmembrane domain, and one or more intracellular activation domains.
In a preferred embodiment, the signal peptide consist of CD8α of SEQ ID NO: 3, the hinge domain consists of CD8α of SEQ ID NO: 4, the transmembrane domain consists of CD8α of SEQ ID NO: 5 or NKGD2 of SEQ ID NO: 6, and the intracellular activation domain consists of CD137/4-1BB of SEQ ID NO: 7, CD28 of SEQ ID NO: 8, CD244/2B4 of SEQ ID NO: 9 and/or of CD3ζ (zeta) of SEQ ID NO: 10.
In a preferred embodiment, the CAR of the invention comprises or consists of SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13. So, three versions of the CAR of the invention have been designed in the context of the present invention:
The second embodiment of the present invention refers to a nucleic acid encoding the CAR of the invention.
In a preferred embodiment, the nucleic acid comprises or consists of SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.
The third embodiment of the present invention refers to cells (hereinafter cells of the invention) comprising the above defined CAR of the invention.
In a preferred embodiment, the cell of the invention is a human T-cell or a human NK-cell.
In a preferred embodiment, the human T-cell is an autologous Tαβ-cell or an allogeneic Tγδ-cell, and the human NK-cell is an allogeneic cord blood-derived cell, a leukemic NK cell line-derived NK cell or an iPS-derived NK-cell.
The fourth embodiment of the present invention refers to a pharmaceutical composition (hereinafter pharmaceutical composition of the invention) comprising a plurality of cells of the invention and, optionally, a pharmaceutically acceptable carrier or diluents.
In a preferred embodiment, the pharmaceutical composition of the invention comprises a plurality of cells of the invention and, optionally, saline plus 2.5% human albumin as pharmaceutically acceptable carrier or diluent.
The fifth embodiment of the present invention refers to the pharmaceutical composition of the invention for use as a medicament, preferably for use in the treatment of invasive fungal infections, more preferably for use in the treatment of invasive fungal infections comprising fungal species from the genus Candida, Aspergillus, Fusarium, Cryptococcus, Paracoccidioides, Histoplasma, Rizhopus, Mucor or Arthrocladium.
The sixth embodiment of the present invention refers to a method for treating invasive fungal infections, more preferably invasive fungal infections comprising fungal species from the genus Candida, Aspergillus, Fusarium, Cryptococcus, Paracoccidioides, Histoplasma, Rizhopus, Mucor or Arthrocladium, which comprises the administration of a therapeutically effective amount of the pharmaceutical composition or the cells of the invention.
For the purpose of the present invention the following terms are defined:
The present invention is illustrated by means of the Examples set below without the intention of limiting its scope of protection.
Immunodeficient NSG (NOD/SCID IL-2Rγcnull) mice from Charles River Laboratories (France) were bred and kept in individual ventilated cages under specific pathogen-free (SPF) conditions. Unless otherwise stated, animals of 8 to 12 weeks of age were used in all experimental procedures, which were approved by the Animal Experimentation Ethical Committee of the University of Barcelona and Generalitat de Catalunya.
The CD5CAR-pCCL lentiviral expression vector of the invention was generated by replacing the CD19-scFv coding sequence from the third-generation lentiviral vector pCCLsinPPT_EFla_CART19 vector (Milone et al., 2009; Porter et al., 2011; Castella et al., 2019) by SEQ ID NO: 11 as a MluI-BspEI fragment. The resulting CD5CAR construct is under the transcriptional control of the EF1α promoter and contains the coding sequences for the CD8α signal peptide (SEQ ID NO: 3), the CD5 extracellular sequence from position R25 to D369 (SEQ ID NO: 1), the CD8α hinge (SEQ ID NO: 4) and transmembrane region (SEQ ID NO: 5), and the intracytoplasmic activating motifs of the 4-1BB/CD137 (SEQ ID NO: 7) and CD35 (SEQ ID NO: 10) receptors. Alternative CD5CAR constructs (SEQ ID NO: 12 and SEQ ID NO: 13) were generated by combining the NKG2D transmembrane region (SEQ ID NO: 6), and the CD28 (SEQ ID NO: 8) and CD244/2B4 by (SEQ ID NO: 9) intracytoplasmic activating motifs.
HEK 293T cells (9×106 cells) were plated into 10-cm culture dishes 24 h before co-transfection with the CD5CAR-pCCL vector and the packaging plasmids pRSV-Rev (Addgene, 12253), and pMDLg-pRRE (Addgene, 12251) and the envelope plasmid pMD2.G (Addgene, 12259). At the transfection time, 16 μg of total DNA (8 μg pCCL-CD5CAR, 4 μg pMDLg/pRRE, 2 μg pRSV-Rev, and 2 μg pMD2.G) were diluted in serum-free DMEM (41966-052; GIBCO) and then mixed with 40 μg of linear polyethylenimine (PEI) (23966-1; Polysciences) for 20 min at room temperature (RT). Next, medium was replaced, and DNA-PEI complexes were added into the cells. At 4 h post transfection, medium was replaced again. Complete lentiviral particles were collected from culture supernatants 72 and 96 h later, passed through 0.45 μm filters and concentrated by centrifugation at 18000 rpm for 3 h at 4° C. Lentivirus-containing pellets were suspended in DMEM and stored at −80° C. until used. Whole lentiviral particles from packaging HEK 293T cells were used to transduce activated peripheral blood human T cells, cord blood-derived NK cells (CBNKs), or leukemia-derived NK cells (NK-92 or KHYG-1 cell lines), which were then subjected to different in vitro and in vivo experimental procedures.
Buffy-coats and cord blood samples from healthy donors were provided by the Banc de Sang i Texits (BST) of Generalitat de Catalunya upon protocols approval by the internal Ethical Committee. For isolation of human T cells, buffy coats were subjected to density centrifugation over Ficoll (10771-6×100ML; Sigma-Aldrich) for isolation of peripheral blood mononuclear cells (PBMCs). Upon monocyte removal by adherence to plastic for 2 h at 37° C. in RPMI 1640 medium with L-glutamine supplemented with 10% FBS, 100 UI/mL penicillin (6191309; Lab EBN), and 100 μg/mL streptomycin (624569; Lab Normon), non-adherent PBMCs (1×106 cells/mL) cells were then activated and expanded for 72 h with Dynabeads™ Human T-Activator CD3/CD28 (111-32D; GIBCO) (at a bead to cell ratio of 1:1) in the same RPMI 1640 medium as above further supplemented with 50 μM β-mercaptoethanol (31350-010; Thermo Fisher) and 30 IU/mL IL-2 (11011456001; Roche). For isolation of human CBNKs, cord blood samples were subjected to magnetic depletion with the NK cell Isolation Kit (MiltenyiBiotec, San Diego, CA) following the manufacturer's instructions. CBNK cells were then expanded for 14 days by co-culture with K562-based antigen presenting cells expressing membrane bound IL-21 (clone 9.mbIL21) in 45% RPMI-1640 and 45% Click's media (Irvine Scientific, Santa Ana, CA) plus 10% human AB plasma (Atlanta Biologicals, Lawrenceville, GA) and IL-2 (400 IU/m added every other day; Proleukin, Chiron, Emeryville, CA).
The human NK cell line NK-92 was obtained from American Type Culture Collection (ATCC, CRL-2407). The human NK cell line KHYG1 was kindly provided by Michael O'Dwyer (ONK Therapeutics, Galway, Ireland). NK-92 cells were cultured in X-VIVO 10 medium (H3BEBP04-743Q, Lonza) supplemented with 5% human AB plasma and 10 ng/ml IL-2 (200-02, Peprotech). KHYG-1 cells were cultured in RMPI 1640 medium with L-glutamine and supplemented with 10% FBS, 100 UI/mL penicillin, 100 μg/mL streptomycin and 10 ng/ml IL-2 (200-02, Peprotech).
Lentiviral transduction of T, CBNK, NK-92 or KHYG-1 cells (1×106) was performed by centrifugation (2000 rpm) for 90 min at 32° C. followed by 5 h-incubation at 37° C. in a 5% CO2 atmosphere, in the presence of 4 μg/mL of polybrene (TR-1003-G; Merck Millipore) in the corresponding growth media for each cell type. Next, fresh medium was added to avoid polybrene toxicity and left in culture for 72 h for further cell surface CD5CAR expression analysis by flow cytometry. To this end, CD5CAR-transduced and untransduced cell controls were adjusted to the desired concentration (1×106 cells/mL) in staining solution (PBS plus 2% FBS) plus PercPCy5.5-labelled anti-human CD5 monoclonal antibody (mAb, clone UCHT2; TONBO) and incubated for 20 min at 4° C. in dark. Next, cells were centrifuged at 1500 r.p.m., washed twice with PBS and fixed in PBS plus 1% paraformaldehyde (PFA) for analysis in a BD FACSCanto II flow cytometer. Mean fluorescence intensity (MFI) or percentage of positive cells was assessed using FlowJo software (Tree Star, USA).
All functional assays with CD5CAR-T/NK cells and the corresponding untransduced cells were performed at 72 h post lentiviral transduction. CD5CAR-transduced and untransduced T/NK cells were co-cultured for 4 h at 37° C. in a 5% CO2 atmosphere with alive C. albicans, C. neoformans or F. solani at different effector:target (E:T) ratios. Next, cells were subjected to osmotic lysis and supernatants seeded on Sabouraud dextrose agar plates for 48 h at 30° C. for CFUs counting. Killing percentage was calculated with regard to fungal cells cultured alone. In parallel experiments, the expression of the T/NK cell degranulation marker CD107a/LAMP1 was assessed by staining with PE-labelled H4A3 mAb (BD Pharmigen) and further analysis of MFI or percentage of positive cells by flow cytometry. The concentration of IFN-γ in supernatants from 24 h co-cultures (in the presence or absence of recombinant soluble human CD5 protein; 10 μg/mL) was assessed by ELISA (BD OptEIA-Human ELISA Set) following the manufacturer's instructions.
C. albicans (strain SC5314; ATCC MYA-2876), kindly provided by Dr. Oscar Zaragoza (Instituto de Salud Carlos III, Madrid), was grown for 48 h at 30° C. on Sabouraud agar plates (01024_00; Conda). Then, a single colony was grown overnight (o/n) at 37° C. in Sabouraud liquid medium (CM0147; Oxoid) under horizontal shaking at 180 rpm. The desired concentration for inoculum preparation was achieved after plating serial dilutions in saline. Infection of NSG mice was performed by intravenous (i.v.; tail vein) injection of 3×104−3×102 colony forming units (CFUs) per gram. Survival and body weight loss were monitored daily.
With the aim of developing a therapeutic strategy against systemic fungal infections, human T and NK cells expressing an activating CD5-based chimerical receptor (CD5CAR) were generated for further adoptive cell transfer therapeutic purposes. To this end, a second generation CD5CAR construction was designed composed of the CD8α signal peptide, the human CD5 extracellular ectodomain (from Arg25 to Asp369), the CD8α transmembrane region and the intracytoplasmic activating domains of 4-1BB/CD137 and CD35 receptors (
CD5CAR-transduced peripheral blood human T cells were co-cultured for 4 h with alive C. albicans or C. neoformans conidia at different effector:target (E:T) ratios. As illustrated by
Similar results regarding higher percentage of C. albicans killing and IFN-g production were observed when CD5CAR-CBNK cells were co-cultured for 4 h with alive C. albicans at different E:T ratios (
Fungal killing experiments in which alive C. albicans, C. auris, C. neoformans and F. solani were co-cultured for 4 h with CD5CAR-transduced or untransduced NK-92 or KHYG1 cells at different E:T ratios demonstrated the higher killing capability of the former cells against different fungal species, thus confirming their broad anti-fungal activity (
The in vivo efficacy of adoptively transferred CD5CAR-T cells and untransduced human T cells to C. albicans-infected immunodeficient NSG mice was next investigated. To that end, a lethal inoculum of C. albicans (8×103 CFU/mouse/gr; as determined in previous dose-course experiments) was i.v. injected to NSG mice. Then, mice were adoptively transferred with CD5CAR-T or untransduced human T cells (4×106 cells/mouse, i.v.) at 24 h post-infection. As illustrated by
Similar in vivo efficacy results were observed when C. albicans-infected NSG mice were adoptively transferred at 24 h post-infection with CD5CAR-CBNK (2×106 cells/mouse, i.v.) and CD5CAR-NK92, (2×106 cells/mouse, i.v.) compared to their untransduced cell controls (
In conclusion, the results provide first proof of concept on the feasibility of CD5-based adoptive T/NK cell therapies in invasive infection by different fungal species.
59-year-old patient diagnosed with acute panmyelosis with myelofibrosis. He received induction chemotherapy with idarubicin and high-dose cytarabine and later consolidation with cytarabine. Experienced relapsed and started treatment with venetoclax plus azacytidine, with partial response after the first two cycles. Last bone marrow aspirate showed 4% of blasts. He had a suitable unrelated donor available and sequential allogenic hematopoietic stem-cell transplant (HSCT) was planned in one month. Primary antifungal prophylaxis with isavuconazole was started due to profound prolonged neutropenia.
The patient was admitted to the hospital for fever, muscle pain, and polytopic erythematous plaques/nodules on the trunk and extremities, some of them with central pustules. Blood tests showed the already known pancytopenia with a marked elevation of C-reactive protein (22 mg/dL). Blood cultures were negative. Galactomannan and β-D-glucan were negative. Chest CT showed multiple peri-bronchial solid nodular images predominantly in the right upper lobe. Bronchoscopy was performed but all results were negative. Skin biopsy was performed and microbiological and anatomopathological studies showed presence of a mold infection, with molecular identification of Fusarium petroliphilum. Antifungal resistance tests showed high minimum inhibitory concentrations (MIC>8 mg/L) for all antifungals tested (voriconazole, isavuconazole, posaconazole, echinocandins, liposomal amphotericin-B), although no clinical breakpoints are established.
Since admission, liposomal amphotericin-B was initiated and after fusariosis diagnosis, terbinafine and voriconazole were added. Despite this treatment, the patient remained febrile, with appearance of new skin lesions and mild worsening of the chest CT images. In this context, compassionate use of the cells of the invention (CD5CAR-NK929) was approved by the Spanish Drug Agency (AEMPS).
After a 72 hours of expansion process of the cells, first-in-human use of irradiated (10 Gy) CD5CAR-NK92 cells was initiated by day +7 since admission. Increasing cell doses (from 7×106 to 1000×106 cells) were administered at 2 to 5 day intervals (see
Despite the relatively preserved general condition, the baseline haematological disease clearly progressed with the appearance of increasing blasts in blood. In this context, due to the lack of available therapies for the haematological malignancy in that context, all therapies were stopped, and comfort was prioritized. The patient died on day +36. Autopsy was denied by the family members.
So, in conclusion, although the patient finally died due to the baseline haematological disease, it can be affirmed that the therapy based on the administration of the cells of the invention (CD5CAR-NK929) was successful in the treatment of the fungal infection.
| Number | Date | Country | Kind |
|---|---|---|---|
| 21382593.8 | Jul 2021 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/068416 | 7/4/2022 | WO |
