[go: up one dir, main page]

WO2020127277A1 - Cellule présentatrice du cmh-i chargée de peptides et il-2 présentant une demi-vie prolongée pour amplifier une réponse immunitaire cytotoxique cellulaire - Google Patents

Cellule présentatrice du cmh-i chargée de peptides et il-2 présentant une demi-vie prolongée pour amplifier une réponse immunitaire cytotoxique cellulaire Download PDF

Info

Publication number
WO2020127277A1
WO2020127277A1 PCT/EP2019/085667 EP2019085667W WO2020127277A1 WO 2020127277 A1 WO2020127277 A1 WO 2020127277A1 EP 2019085667 W EP2019085667 W EP 2019085667W WO 2020127277 A1 WO2020127277 A1 WO 2020127277A1
Authority
WO
WIPO (PCT)
Prior art keywords
hll
antigen
cells
cell
peptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2019/085667
Other languages
English (en)
Inventor
Richard Kroczek
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Robert Koch Institute
Original Assignee
Robert Koch Institute
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Robert Koch Institute filed Critical Robert Koch Institute
Publication of WO2020127277A1 publication Critical patent/WO2020127277A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • A61K2039/55527—Interleukins
    • A61K2039/55533—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/605—MHC molecules or ligands thereof

Definitions

  • the present invention relates to a peptide-loaded MHC-I presenting cell and an IL-2 with extended half-life for use in amplifying a cellular cytotoxic immune response and related methods.
  • the main function of the immune system is to protect the host against invading pathogens, including bacteria, viruses, parasites and others.
  • the so-called “adaptive” branch of the immune system consists of B cells, which produce antibodies, and CD4 + and CD8 + T cells.
  • CD4 + T cells One function of CD4 + T cells is to instruct (“help”) B cells to generate certain type of antibodies and to help CD8 + T cells to differentiate into specialized effector cells. Therefore, these CD4 + T cells are also called T helper (Th) T cells.
  • Another subset of CD4 + T cells are regulatory T cells (Treg) which suppress adaptive T cell responses.
  • the immune system discriminates harmless foreign antigens such as proteins present in ingested food, from “dangerous” foreign antigens such as proteins originating from invading pathogens.
  • This discrimination is made possible by structures which are highly specific for pathogens (“pathogen-associated molecular patterns”, PAMPs).
  • PAMPs pathogen-associated molecular patterns
  • LPS lipopolysaccharide
  • ds double-stranded
  • RNA of viruses Skoberne et al. 2004, Silva-Gomes et al. 2014.
  • APC professional antigen presenting cells
  • DC dendritic cells
  • a microbial pathogen containing a PAMP is phagocytosed by an APC, particularly by a dendritic cell (DC)
  • the PAMP is sensed by the APC with the corresponding Pattern Recognition Receptor (PRR) and is thus recognized as “danger”.
  • PRR Pattern Recognition Receptor
  • DC process the protein component of the pathogen, and migrate to the lymph nodes, where they present the processed microbial protein to CD4 + and CD8 + T cells in the context of the MHC.
  • the T cells become activated and mount an adaptive response suited (“polarized”) to effectively combat the type of microbial threat (Netea et al. 2005, Mittmcker et al. 2014).
  • the response of the immune system to an invading pathogen depends on the nature of the associated PAMP. Intracellular pathogens cannot be eliminated by antibodies and require a Th1 response for clearance. In a Th1 response, polarized CD4 + T cells secrete a set of cytokines, among them the Th1 signature cytokine IFN Y, CD8 + T cells differentiate into cytotoxic T cells (Tc1 cells) capable of killing the cells harboring the intracellular pathogen. It is therefore not surprising that various PRR in the APC responsible for sensing intracellular pathogens, such as viruses, trigger a rather uniform Th1/Tc1 immune response in which CD4 + T cells generate IFN-g and CD8 + T cells differentiate into cytotoxic cells capable of eliminating the infected cells.
  • the goal is to induce a Th1/Tc1 immune response against a given tumor antigen or microbial antigen.
  • the host can be immunized in a variety of ways. This can be done by vaccination with life or attenuated viruses, and life or attenuated bacteria: the DC recognize the PAMP of the viral (e.g. dsRNA) or bacterial pathogen and a corresponding Th1/Tc1 response is mounted.
  • a therapeutic vaccination can also be also done with nucleic acid preparations (RNA or DNA) coding for antigenic proteins. These nucleic acid-based vaccines differ from host RNA and DNA and are recognized by the PRR machinery of the APC as foreign (“dangerous”) DNA or RNA, and a corresponding Th1/Tc1 response ensues; the danger signal comes from the nucleic acid itself - these types of vaccines are therefore called“self- adjuvanted”.
  • Th1/Tc1 response In cases of DNA or RNA vaccines which do not trigger a“danger” signal through a PRR and with peptide or protein vaccines which do not contain any PAMP, no Th1/Tc1 response is induced. In these cases, however, the PAMP- sensing machinery of the immune system can be triggered by co-administration of known adjuvants which either represent a Th1 danger signal or which mimick a Th1 danger signal. Immunization with a peptide or protein in combination with an adjuvant which provides a Th1/Tc1 danger signal (“Th1 adjuvant which is a danger signal”), uniformly results in the induction of Th1/CD8 T cell cytotoxic immunity against the employed antigen (WO 2015/140172, Flartung et al. 2015).
  • Therapeutic vaccines can be applied in a non-targeted fashion by injection into the skin or subcutaneously (s.c.).
  • the vaccine can be targeted to professional APC such as macrophages, B cells, and DC (Example 6 of WO 2015/140172).
  • APC such as macrophages, B cells, and DC
  • the most effective target are DC.
  • XCR1 + DC are specialized on the uptake of dead cell material and its presentation in the context of MHC I to CD8 + T cells, the correspondent DC in the human are the (XCR1 + ) CD141 + (BDCA3 + ) DC (van Montfoort et al. 2014, Gurka et al. 2015).
  • SIRPa + DC in the mouse and the human are specialized on the uptake of soluble material and its presentation in the context of MHC II to CD4 + T cells (Gurka et al. 2015).
  • skin DC such as Langerhans’ cells, monocyte- derived DC, and plasmacytoid DC (Merad et al. 2013).
  • None of the functions of DC subpopulations appear to be unique and exclusive, e.g. also SIRPa + DC can present antigen to CD8 + T cells (Bachem et al. 2012, Example 6 of WO 2015/140172), albeit suboptimally.
  • other APC e.g.
  • macrophages can transfer proteins to XCR1 + DC, which then degrade the proteins and present the derived peptides to CD8 + T cells (Backer et al. 2010). All of these APC can be targeted with a therapeutic vaccine. When this is done with an appropriate Th1 adjuvant which is a danger signal, this will uniformly result in induction of cytotoxic CD8 + T cells.
  • the vaccine antigen has to enter an APC or (in case of nucleic acids) has to be translated into a protein which ends up in the APC.
  • the proteinaceous antigen is then processed and presented in the context of MHC-I and MHC-II molecules on the APC surface to CD8 + T cells and CD4 + T cells, respectively.
  • the modes of immunization or antigen delivery may differ, the outcome is uniform and results in the activation of T cells by antigen-derived peptides presented by the APC. If the presenting APC at the same time senses a Th1 -danger signal through an Th1 adjuvant which is a danger signal (e.g.
  • EBV chronically infected individuals
  • T cell therapy various approaches were developed in order to activate and expand antigen-specific CD8 + T cells in vitro and re-infuse them into the patients (“adoptive T cell therapy”).
  • These can be natural antigen-specific T cells isolated from the patient or T cell equipped with an engineered T cell receptor (Rosenberg et al., 2015).
  • an engineered T cell receptor Rost et al., 2015.
  • these natural or engineered T cell populations tend to loose their activity and to die. Therefore, there is a need to re activate and expand those adoptively transferred T cell populations in vivo.
  • IL-2cx has been shown to be clearly superior in expanding CD8 + T cells in vivo in comparison to another compound with prolonged half-life, in which IL-2 (capable of binding to CD25) has been fused to an antibody (Fig. 4 in Letourneau et al., 2010); ii) the antibody in the IL-2cx used for our amplification procedure has been shown to prevent the binding of the complex to the high affinity IL-2 receptor, the CD25 molecule, and thus to prevent many unwanted effects, including activation of regulatory T cells (T reg ) expressing high levels of CD25 (Boyman et al., 2006, Krieg et al. , 2010). Due to their high dependency on CD25-mediated signals for survival, T reg cells are good indicators for unwanted, CD25-mediated effects.
  • IL-2 fused to an antibody to immunize mice.
  • this construct apparently similar to the one used by Letourneau et al., 2010, was employed by chance after application of ADAS, we surprisingly discovered that this form of IL-2 is as potent as IL-2cx for the expansion of CD8 + T cells after ADAS (Example 3 and Fig. 3).
  • IL-2ext extended half-life
  • IL-2ext offers major advantages over IL-2cx in regards to manufacturing (only one compound has to be produced), formulation (formulation is less complicated), stability (the covalently attached IL-2 cannot dissociate), and regulatory requirements (evidence for integrity of the complex over time is not an issue).
  • a hlL-2 variant with extended half life (IL-2ext) together with ADAS for amplification of CD8 + T cell responses in vivo is a major step forward in the treatment of patients with tumors or chronic infections. It was surprisingly found that amplification is efficient when the administration of the hlL-2 variant begins within 5 days after the administration of ADAS, and when the h IL-2 variant is administered at least once daily or continuously, and is administered for at least 2 subsequent days (Example 4 and Fig. 4).
  • an efficient amplification of a cellular cytotoxic immune response against an antigen-comprising protein in a patient can be achieved by administering a peptide-loaded MHC-I presenting cell to a patient in step i), followed in step ii) by the administration of a hlL-2 variant with extended circulating half-life in vivo in a human as compared to hlL-2, wherein the hlL-2 variant is capable of binding to the high affinity IL-2 receptor chain (CD25), wherein the administration of the h IL-2 variant of step ii) begins within 5 days after the administration of the peptide-loaded MHC-I presenting cell of step i), and wherein the hlL-2 variant is administered at least once daily or continuously, and is administered for at least 2 subsequent days (Example 4 and Fig. 4).
  • administering a peptide-loaded MHC-I presenting cells and IL-2ext amplifies the cytotoxic immune response from day 4 after priming of the patient’s CD8 + T cells with antigen to day 29, after which the CD8 + T cells become memory cells (Example 5 and Fig. 5).
  • the present invention relates to a medicament for use in a method of amplifying a cellular cytotoxic immune response against an antigen-comprising protein in a patient, the method comprising the steps of:
  • the peptide is derived from the antigen-comprising protein, thereby re-activating the activated T cell, wherein the peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell is administered only once and in a time frame of from 4 days to 29 days after the T cells were activated against an antigen, and
  • MHC-I major histocompatibility complex class I
  • hlL-2 variant administered at least once daily or continuously, and is administered for at least 2 subsequent days.
  • the present invention relates to a medicament for use in a method of amplifying a cellular cytotoxic immune response against an antigen-comprising protein in a patient, the method comprising the steps of: i) administering to a patient having T cells activated against an antigen a peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell and a Th1 adjuvant which is a danger signal,
  • MHC-I major histocompatibility complex class I
  • the peptide is derived from the antigen-comprising protein, thereby re-activating the activated T cell, wherein the peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell is administered only once and in a time frame of from 4 days to 29 days after the T cells were activated against an antigen, and
  • MHC-I major histocompatibility complex class I
  • hlL-2 variant administered at least once daily or continuously, and is administered for at least 2 subsequent days.
  • any mode of primary activation (“priming”) of the patient’s CD8 + T cells to cytotoxic cells may be used for achieving amplification with the combination of the peptide- loaded major histocompatibility complex class I (MHC-I) presenting cells and a hlL-2 variant.
  • Priming can be achieved by non-targeted vaccination or vaccination targeted to APC employing a Th1 adjuvant which is a danger signal (Example 1 and Fig. 1 ).
  • the vaccination can be nucleic-acid based (RNA, DNA) or protein- based and can employ microbe-derived vectors, and can also be self-adjuvanted.
  • adoptively transferred antigen- specific CD8 + T cells may also be amplified using a peptide-loaded major histo compatibility complex class I (MHC-I) presenting cell and a Th1 adjuvant which is a danger signal, within a time-frame of 4 to 29 days after last activation in vitro before transfer.
  • MHC-I major histo compatibility complex class I
  • a hlL-2 variant with extended circulating half-life in vivo in a human as compared to hlL-2 wherein the hlL-2 variant is capable of binding to the high affinity IL-2 receptor chain (CD25), wherein the administration of the h IL-2 variant of step ii) begins within 5 days after the administration of the peptide-loaded MHC-I presenting cell of step i), and wherein the hlL-2 variant is administered at least once daily or continuously, and is administered for at least 2 subsequent days (Example 7 and Fig. 7).
  • adoptively transferred antigen-specific CD8 + T cells may also be amplified using a peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell and a Th1 adjuvant which is a danger signal, administered more than 29 days after last activation in vitro before transfer and/or after the activated cytotoxic T cells have returned to the memory state and/or when a patient has at least one memory CD8+ T cell or memory T cell against said antigen.
  • MHC-I major histocompatibility complex class I
  • a hlL-2 variant with extended circulating half-life in vivo in a human as compared to hlL-2 wherein the hlL-2 variant is capable of binding to the high affinity IL-2 receptor chain (CD25), wherein the administration of the hlL-2 variant of step ii) begins within 5 days after the administration of the peptide-loaded MHC-I presenting cell of step i), and wherein the hlL-2 variant is administered at least once daily or continuously, and is administered for at least 2 subsequent days.
  • the present invention relates to a medicament for use in a method of amplifying a cellular cytotoxic immune response against an antigen-comprising protein in a patient, the method comprising the steps of:
  • step (x) administering to the patient of step (x) a peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell and a Th1 adjuvant which is a danger signal,
  • MHC-I major histocompatibility complex class I
  • the peptide is derived from the antigen-comprising protein, thereby re-activating the activated T cell(s), wherein the peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell is administered only once and is administered in a time frame of from 4 days to 29 days after the one or more T cell(s) administered to the patient in step (x) were activated against an antigen, and
  • MHC-I major histocompatibility complex class I
  • hlL-2 variant administered at least once daily or continuously, and is administered for at least 2 subsequent days.
  • the present invention relates to a medicament for use in a method of amplifying a cellular cytotoxic immune response against an antigen-comprising protein in a patient, the method comprising the steps of:
  • step (x) administering to the patient of step (x) a peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell and a Th1 adjuvant which is a danger signal,
  • MHC-I major histocompatibility complex class I
  • the peptide is derived from the antigen-comprising protein, thereby re-activating the activated CD8 + T cell(s), wherein the peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell is administered only once and is administered in a time frame of from 4 days to 29 days after the one or more T cell(s) administered to the patient in step (x) were activated against an antigen, and
  • MHC-I major histocompatibility complex class I
  • hlL-2 variant administered at least once daily or continuously, and is administered for at least 2 subsequent days.
  • autologous antigen-specific T cells can be used.
  • antigen-specific MHC-I restricted T cells induced by an infection or by a tumor can be isolated from a patient either from the peripheral blood or from tumor tissue.
  • These autologous antigen-specific T cells then can be activated in vitro in an antigen-specific manner by standard techniques known in the art, such as adding peptides or proteins to a culture containing APC, or using a suitable MHC-I presenting cell line and growth factors.
  • the peptide-loaded MHC-I used for stimulation in vitro can be synthetic.
  • the activated, autologous MHC-I restricted T cells can be expanded in vitro and are subsequently re-infused into the patient.
  • Autologous cells or cells from a different individual capable of cytotoxic function can, in a yet further preferred embodiment, be engineered to express a natural or chimeric T cell receptor (TCR).
  • TCR T cell receptor
  • the natural TCR (a-and b-chains) can be isolated from MHC-I restricted T cells with known antigen specificity present in the peripheral blood or tumor tissues of an individual (e.g. from T cells recognizing either a neoantigenic peptide or an antigenic peptide from a shared antigen, such as NY-ESO-1 , or a peptide from an infectious agent).
  • autologous cells or cells from a different individual can be engineered with chimeric TCRs with known antigen-specificity, in which the antigen-recognizing extracellular portions of the TCR (a-and b-chains) are combined with signaling elements amplifying the TCR signal, a principle currently used for CAR-T cells (Walseng et al. , 2017).
  • MHC-I restricted T cells which may or may not express CD8 (which is not absolutely required for recognition of MHC-I restricted peptides by the T cell receptor), are activated and expanded in vitro and are then infused into the patient for adoptive T cell transfer.
  • CD8 which is not absolutely required for recognition of MHC-I restricted peptides by the T cell receptor
  • the MHC-I restricted cells will become activated and exert effector functions in vivo, including cytokine secretion and cytotoxicity, similar to natural MHC-I restricted T cells. In vivo, they can also be re activated by autologous cells loaded with the corresponding antigenic peptide and a Th1 adjuvant which is a danger signal. If not further activated, they become memory T cells.
  • MHC-I restricted T cells are selected from antigen-specific, MHC-I restricted T cells from blood or from tumor-infiltrating lymphocytes, or from T cells, NK cells, or NK T cells genetically engineered with natural or chimeric MHC-I restricted T cell receptors of known specificity, and/or are autologous or allogenic T cell(s), which are optionally genetically modified MHC-I restricted T cell(s).
  • the activated T cells are MHC-I restricted T cells.
  • step i) administering to a patient having T cells activated against an antigen a peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell and a Th1 adjuvant which is a danger signal
  • the peptide-loaded MHC-I presenting cell is administered to the patient in a time frame of from 4 days to 29 days after initial“priming” with the antigen, whereby the T cells are activated against an antigen.
  • T cells are activated against an antigen.
  • Targeted as well as non-targeted approaches for“priming” and thereby activating the T cells are available.
  • the peptide-loaded MHC-I presenting cell is administered to the patient in a time frame of from 4 days to 29 days after the one or more MHC-I- restricted T cell(s) or cells with an engineered T cell receptor (natural or chimeric TCR) were activated in vitro for the last time.
  • the peptide-loaded MHC-I presenting cell is administered to the patient more than 29 days after the one or more MHC-I restricted T cell(s) or cells with an engineered T cell receptor (natural or chimeric TCR) were activated in vitro for the last time and/or after the activated cytotoxic T cells have returned to the memory state and/or when a patient has at least one memory MHC-I restricted T cell or memory T cell against said antigen.
  • an engineered T cell receptor naturally or chimeric TCR
  • peptide-loaded MHC-I presenting cell is administered only once to the patient within the indicated time frame for the above embodiments of the invention.
  • Primer is understood as inducing a primary cellular cytotoxic immune response to a given antigen. Such priming step is typically induced in vivo in the patient.
  • administering one or more T cell(s) activated against an antigen to the patient is understood as an adoptive transfer of various antigen-specific, MHC-I restricted T cell populations, which may or may not express CD8.
  • These can be natural antigen-specific T cells isolated from the patient by known techniques (e.g. tetramer-based isolation) and then activated and expanded in vitro.
  • these can be autologous or allogenic cells (T cells, NK cells, or NKT cells) equippped with an engineered natural or chimeric T cell receptor of known antigen-specificity and then activated in vitro before transfer.
  • the peptide is derived from the antigen-comprising protein” is understood as that the peptide sequence is part of the antigen-comprising protein sequence; i.e. is a subsequence of antigen-comprising protein sequence.
  • the peptide comprises the antigen(s) or epitope(s) of interest.
  • A“peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell” is understood as a cell which presents desired peptides on the surface of the cell by binding to MHC-I.
  • an “antigen-comprising protein” is understood as protein which comprises an antigen or epitope of interest. Accordingly, in a preferred embodiment, an antigen comprising protein is a protein which comprises a peptide sequence, which may be continuous or discontinuous, which is recognized as antigen in the context of MHC-I by a corresponding T cell receptor (TCR).
  • TCR T cell receptor
  • a “cellular cytotoxic immune response” is understood as Th1 -type cellular cytotoxic immune reaction or Th1/Tc1 immune response that can be elicited to a given antigen. This is in contrast to the response to classical vaccines which mainly address the Th2 antigen presentation pathway and mainly lead to the generation of Th2-type (neutralizing) antibodies and immune reactions.
  • An“amplified” a cellular cytotoxic immune response is understood as a cellular cytotoxic immune response which occurs for a longer time and/or more strongly as compared to the cellular cytotoxic immune response obtained by priming with an targeted or non-targeted antigen, as described herein for the methods of activating T cells against an antigen-comprising protein or to the effects achieved with adoptive transfer of cytotoxic T cells alone.
  • the response may be extended by 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or more days and/or the effect at a given time point is amplified.
  • an amplified cellular cytotoxic immune response is understood as a cellular cytotoxic immune response which is in addition amplified compared to the cellular cytotoxic immune response obtained by the“priming” with an antigen, as described below.
  • the response may be characterized by 2-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, or 20-fold or more amplification of CD8 + T cells specific for the antigen as compared to CD8 + T cells specific for the antigen present at a given time point after priming the patient with the antigen.
  • the response may be characterized by 100-fold or more, 1000-fold or more, 100.000-fold or more, or 10 6 -fold or more amplification of CD8 + T cells specific for the antigen as compared to naive CD8 + T cells specific for the antigen.
  • Methods for determining the CD8 + T cells specific for the antigen are known to a skilled person and are described in the Examples.
  • an efficient amplification of the cellular cytotoxic immune response is achieved by administration of the peptide-loaded MFIC-I presenting cell in step i) after the activated cytotoxic T cells have returned to the memory state and/or when a patient has at least one memory CD8 + T cell or memory T cell against said antigen.
  • T cells have returned to the memory state “Memory T cells” or“Memory CD8 + T cells” are understood as T cells which were already at least once activated in vivo or in vitro more than 29 days ago and then have returned to a dormant state.
  • Memory CD8 + T cells or“Memory T cells” are known to the skilled person and are understood as T cells, which are initially CD8 + T cells, that have recognized a specific antigen and have in the past responded to said antigen. Memory cells are dormant and do not exert effector function, but they can respond more quickly and with greater strength to a re-challenge by the same antigen. The person skilled in the art is able to distinguish naive T cells from recently activated,“primed” T cells, and recently activated, “primed” T cells from memory T cells using a variety of surface markers.
  • biomarkers are, for example, CD25, CD45RA, CD45R0, CD62L, CCR7, ICOS, and XCL1 , such as described in Paul W.E. (2013).
  • biomarker pattern differs in various organs.
  • Methods for determining antigen-specific CD8 + T cells are known to a skilled person and are for example described in the Examples.“Memory T cells” are typically CD8 + T cells, however, cases are known where the Memory T cells are no longer express the CD8 molecule.
  • Naive CD8 + T cells can be identified as CD45RA + , CD27 + , CD28 + , CD45RO , CD62L + , CCR7 + , CD127 + , CD132 + cells.
  • Naive CD8 + T cells are understood as cells which have not yet encountered an antigen in vivo, have a high activation threshold and therefore require priming by professional APC, which provide special co-stimulation via CD80 and CD86.
  • CD8 + T cells Upon recognition of antigen, CD8 + T cells become activated, expand, and acquire effector functions, such as secretion of TNF-a IFN-y, including cytotoxic capacity, by expression of granzyme B. Later, the expanded effector T cell population contracts due to apoptosis. However, some of the activated T cells become memory cells, which survive for a long time. Antigen-specific CD8 + T cells detected 30 days after first activation or “priming” are understood as memory CD8 + T cells.
  • CD8 + memory T cells or memory T cells there are several subtype of CD8 + memory T cells or memory T cells: i) Central memory T cells (TCM), found in lymph nodes and the circulation, expressing CD45RO, CCR7, and CD62L; ii) effector memory T cells (T E M and TEMRA cells) expressing CD45RO but lacking expression of CCR7 and CD62L, and are found in the circulation and tissues; iii) stem memory cells (T S CM), like naive T cells are CD45RA + , CD45RO , CCR7 + , CD62L + , CD27 + , CD28 + and IL-7Ra + , but they also express large amounts of CD95, IL-2RP, CXCR3, and LFA-1 (Farber et al., 2014).
  • TCM Central memory T cells
  • T E M and TEMRA cells effector memory T cells
  • T S CM stem memory cells
  • activated cytotoxic CD8 + T cells become memory cells 30 days after the T cells were activated (or“primed”) against the antigen.
  • Memory CD8 + T cells or memory T cells can be re-activated from their dormant state by antigen presented by professional APC, but also by antigen presented by other MHC-l-bearing cells, since they have a lower activation threshold (Paul, Fundamental Immunology 7 th ed., p. 741 ff).
  • the present invention relates to medicament for use in a method of amplifying a cellular cytotoxic immune response against an antigen-comprising protein in a patient, the method comprising the steps of:
  • peptide-loaded major histocompatibility complex class I MHC-I
  • Th1 adjuvant which is a danger signal
  • the peptide is derived from the antigen-comprising protein
  • T cell(s) activated against an antigen at least 30 days before and
  • hlL-2 variant administered at least once daily or continuously, and is administered for at least 2 subsequent days.
  • the present invention relates to a medicament for use in a method of amplifying a cellular cytotoxic immune response against an antigen-comprising protein in a patient, the method comprising the steps of:
  • peptide-loaded major histocompatibility complex class I MHC-I
  • Th1 adjuvant which is a danger signal
  • the peptide is derived from the antigen-comprising protein
  • T cell(s) activated against an antigen at least 30 days before
  • hlL-2 variant administered at least once daily or continuously, and is administered for at least 2 subsequent days.
  • the activated T cells are MHC-I restricted T cells.
  • the infection is a chronic viral, bacterial, or parasitic infection.
  • Such chronic infections persist for a longer time, such for at least 30 days, 60 days, 90 days, 4 months, 6 months or 1 year, or longer.
  • the patient suffers from a tumor disease and the antigen is a non-mutated tumor-specific antigen or a neoantigen.
  • tumor-specific alterations may serve as suitable sources for tumor neoantigens which, by definition, are tumor-specific antigens.
  • tumor-specific antigens which, by definition, are tumor-specific antigens.
  • These can be single nucleotide variants resulting in non-synonymous amino acid substitutions, frame shifts in antigen-coding regions due to insertions or deletions, novel peptide epitopes arising from chromosomal translocations, and tumor-specific posttranslational modifications, such as phosphorylation and deamidation, generating novel, tumor-specific epitopes (Braunlein et al. , 2017).
  • Neoantigens can also be protein sequences resulting from alternative splicing only found in tumor cells or resulting from a different transcription pattern of genes found only in tumors (Hensler et al., 2016).
  • tumor-specific peptides can arise from the monoclonal hypervariable recombined immunoglobulin-coding region (Braunlein et al. , 2017).
  • “Antigen-Dependent Amplification System” or “ADAS” is understood as step i) of the embodiments of present invention above, comprising administering to the patient a peptide-loaded major histocompatibility complex class I (MHC I) presenting cell and an Th1 adjuvant which is a danger signal, wherein the peptide is derived from the antigen-comprising protein.
  • MHC I major histocompatibility complex class I
  • Th1 adjuvant which is a danger signal
  • the activated T cell in the patient is re-activated in case of“priming” with the antigen, or the memory CD8 + T cells or memory T cells are re-activated in case the activated T cell specific for the antigen in the patient has returned to the memory state.
  • various MHC-I presenting lymphocytic populations, splenocytes, B cells, T cells, when externally loaded with a peptide in vitro and injected together with a Th1 adjuvant which is a danger signal are capable of providing the signals necessary to continue the initial activation, expansion and functional differentiation of CD8 + T cells to cytotoxic effector cells (WO 2015/140172).
  • the loading of the MHC-I on the lymphocytic populations with an antigenic peptide can also be achieved by internal loading the MHC-l-bearing cell using whole protein which is taken up, degraded, and presented in the context of MHC-I (WO 2015/140172).
  • proteins bound to a cell-penetrating peptide WO 2015/140172
  • bound to other molecules facilitating the entry of the proteins into the cell e.g. receptor-mediated entry, by transient infection or transduction of the lymphocytic population, by transfection with DNA or RNA vectors, or by any chemical or physical disturbance of the cellular membrane allowing entry of proteins, such as complexation with Ca- phosphate, complexation with PEI, electroporation, mechanical squeezing of the cells, etc.
  • cell populations comprising more than one cell type may be used for loading.
  • Cell populations which are preferably used for ADAS are unseparated human peripheral blood mononuclear cells (PBMC), or compositions comprising T cells and B cells isolated from PBMC.
  • PBMC peripheral blood mononuclear cells
  • the few dendritic cells present in the peripheral blood are not regarded as PBMC cells.
  • cells not suitable for ADAS are dendritic cells, in vitro generated dendritic cells, tumor tissues removed from a patient, or tumor cell lines. The time for administration of ADAS to the patient differs, depending on the immunization status of the patient.
  • ADAS is applied in a time frame of between 4 days and 29 days after primary activation of naive CD8 + T cells in vivo (Example 5 and Fig. 5).
  • a patient having memory CD8 + T cells or memory T cells to a given antigen such as patients suffering from a tumor disease, or suffering from an infection for a longer time (30 days or more), or patients who were adoptively transferred with T cells activated in vitro more than 29 days ago, such as 30 days or more, 25 days or more, 40 days or more, 100 days or more, 6 months or more, or 1 year or more, ADAS (together with an Th1 adjuvant which is a danger signal) can be applied at any time, since memory T cells have a lower activation threshold and can directly be activated by the peptide-loaded MHC-I bearing cell.
  • a patient who already has memory T cells to a given antigen can also be pre-treated by antigen delivery, i.e.
  • ADAS is administered in a time frame between 4 days and 29 days after delivery of the antigen.
  • a patient who was adoptively transferred with T cells activated in vitro more than 29 days ago such as 30 days or more, 25 days or more, 40 days or more, 100 days or more, 6 months or more, or 1 year or more, ADAS, together with an Th1 adjuvant which is a danger signal, is applied at any time, since memory T cells have a lower activation threshold and can directly be activated by the peptide-loaded MHC-I bearing cell.
  • a patient who already has memory T cells to a given antigen can also be pre-treated by antigen delivery, i.e. “priming”, since this patient will still harbor naive CD8 + T cells to this antigen and to other relevant antigens derived from the tumor or infection, respectively. In this latter case of“priming”, ADAS is administered in a time frame between 4 days and 29 days after delivery of the antigen.
  • ADAS cells loaded with peptide may be administered by various routes, preferably by systemic administration, more preferably by intravenous or intraperitoneal administration.
  • the number of administered cells is preferably between 1x10 8 to 100x10 9 cells.
  • the ADAS cells loaded with peptide are administered as suspension of cells in a solution, which is more preferably an aqueous solution and/or buffered solution.
  • the peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell of step i) is administered only once to said patient within the indicated time frame, or is administered only once to said patient in case of re activation of memory T cells.
  • ADAS no further administration of ADAS is performed.
  • a peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell is administered to the patient only once within the indicated time frame and,
  • MHC-I major histocompatibility complex class I
  • MHC-I major histocompatibility complex class I
  • the peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell of step i) is effective when administered in a time frame of from 4 days to 29 days after the T cells were activated against an antigen or after the one or more T cell(s) were administered to the patient in case of adoptive T cell transfer.
  • the time frame for the administration of peptide-loaded cells in step i) is from 4 to 29 days, preferably from 5 days to 20 days, more preferably from 5 days to 15 days, even more preferably from 5 days to 12 days.
  • the administration of ADAS is performed 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days or 29 days after the T cells were activated against the antigen or after the one or more CD8 + T cell(s) were administered to the patient in case of adoptive T cell transfer.
  • a hlL-2 variant with extended circulating half-life in vivo in a human as compared to hlL-2 is administered, wherein the hlL-2 variant is capable of binding to the high affinity IL-2 receptor chain (CD25).
  • a strong amplification of the cytotoxic immune response is achieved, when, following an ADAS procedure, administering to the patient a hlL-2 variant with extended circulating half-life in vivo in a human as compared to hlL-2, wherein the hlL-2 variant is capable of binding to the high affinity IL-2 receptor chain (CD25), wherein the administration of the hlL-2 variant of step ii) begins within 5 days after the administration of the peptide- loaded major histocompatibility complex class I (MHC I) presenting cell of step i), and wherein the hlL-2 variant of step ii) is administered at least once daily or continuously, and is administered for at least 2 subsequent days.
  • MHC I major histocompatibility complex class I
  • the hlL-2 moiety in the hlL2 variant is a mature hlL-2 preferably selected from mature wt hlL-2 and aldesleukin.
  • the hlL-2 variant has a circulating half-life in vivo of at least 2 hours in a human and/or comprises at least one hlL-2 moiety.
  • the hlL-2 variant comprises 1 , 2, 3, 4, 5 or more hlL-2 moieties.
  • IL-2 with extended half-life is also designated “IL-2 with extended half-life”, or“IL-2ext” in the present application.
  • Cytokines are molecules which are known to have a short half-life due to glomerular filtration in the kidney.
  • hlL-2 reportedly has a half-life of of several minutes in vivo in the human (Donohue et al., 1983). It has been previously determined that the half-life of a cytokine can be substantially extended in vivo by attaching it to larger, functionally inert molecule which serves as a “scaffold”.
  • IL-2ext is therefore understood as a molecule comprising a human IL-2 (hlL-2), which is non-covalently or covalently, preferably covalently bound to a scaffold, and shows a half-life in a human patient in vivo of at least 2 hours.
  • IL-2ext of the present invention is capable of binding to the high affinity IL-2 receptor chain CD25.
  • “IL-2ext” binds to both the high-affinity (CD25) IL-2 receptor and the low-affinity (CD122) IL-2 receptor.
  • the human IL-2 (hlL-2) is covalently bound to the scaffold recombinantly or chemically.
  • the hlL-2 moiety in“IL-2ext” is covalently linked to a chemical moiety having a molecular weight of at least 15 kDa.
  • Suitable popular scaffolds for use in the invention are known in the art, and comprise immunoglobulins, comprising complete antibodies, Fc-regions of antibodies, and other antibody formats such as antibodies without Fc-region (Diabodies), single chain Fv (scFv) fusions, Fab-scFv fusions and other antibody- derived formats increasing the half-life of the cytokine (Jazayeri et al. , 2008).
  • Such antibodies merely serve to increase half-life in vivo.
  • the antibody portion preferably does not specifically recognize an epitope of the patient’s body. Accordingly, in a preferred embodiment, such antibodies for use as scaffold do not specifically recognize an epitope of the patient.
  • an “antibody” is understood as protein comprising at least one Ig-like domain, preferably comprising at least one antigen-binding domain.
  • at least one antigen-binding domain comprises a VH and/or a VL domain.
  • the antibody is preferably not capable of specifically binding to or recognizing a human antigen. Such antibodies are known in the art and used in the Examples.
  • IL-2 preferred scaffolds
  • Other preferred scaffolds are large human proteins which are functionally inert, such as human serum albumin.
  • Further preferred scaffolds to which IL-2 can be bound are polymers such as PEG (Veronese FM et al., 2008) or polymer mimetics such as hydrophilic and flexible polypeptide chains as used in the XTEN (Schellenberger et al., 2009) or PASylation technologies (Binder et al. 2017).
  • Further preferred scaffolds increasing the half-life of IL-2 are carbohydrates, such as dextran, polysialic acids, hyaluronic acid, dextrin, or hydroxyethyl starch (Pasut 2014). Accordingly, the hlL-2 variant used in the present invention preferably exhibits an increased molecular weight as compared to hlL-2.
  • the hlL-2 variant has a molecular weight of at least 30 kDa and/or wherein the hlL-2 moiety in the hlL-2 variant is covalently linked to a chemical moiety having a molecular weight of at least 15 kDa.
  • the hlL-2 variant has a molecular weight of at least 40 kDa, 50 kDa, 80 kDa, 100 kDa, or 200 KDa.
  • the molecular weight may be up to about 3000KDa, 2000 KDa or 1000 kDa.
  • the hlL-2 moiety in the hlL-2 variant is covalently linked to a chemical moiety having a molecular weight of at least 25 kDa, 35 kDa, 65 kDa, 85 kDa, or 185 KDa.
  • the molecular weight of the chemical moiety may be up to about 3000 kDa, 2000 KDa or 1000 kDa.
  • the hlL-2 variant is selected from a PEGylated hlL-2, hlL-2 linked to hyaluronic acid and hlL-2 fused to at least one peptide or protein, preferably wherein the hlL-2 fused to a least one peptide or protein is hlL-2 fused to an Fc, hlL-2 fused to XTEN, hlL-2 fused to an immunoglobulin, preferably antibody, which is not capable of specific binding to a human antigen, hlL-2 fused to Transferrin, hlL-2 fused to Albumin, preferably human serum Albumin, hlL-2 fused to PEG, hlL-2 fused to a homoamino acid polymer (FIAP), hlL-2 fused to a proline-alanine-serine (PAS) polymer, hlL-2 fused to a carbohydrate, more preferably selected from dextran,
  • FIAP homoamin
  • the“scaffold” is selected from a PEG moiety, such as PEG50, PEG1000 or PEG2000, XTEN, hyaluronic acid, at least one peptide or protein, an immunoglobulin which is not capable of specific binding to a human antigen, an Fc, Transferrin, Albumin, recombinant PEG, a homoamino acid polymer (FIAP), a proline-alanine-serine (PAS) polymer, carbohydrate, more preferably selected from dextran, polysialic acids, hyaluronic acid, dextrin, and hydroxyethyl starch (HES), and an elastin-like peptide (ELP).
  • a PEG moiety such as PEG50, PEG1000 or PEG2000, XTEN, hyaluronic acid, at least one peptide or protein, an immunoglobulin which is not capable of specific binding to a human antigen, an Fc, Transferrin, Album
  • the hlL-2 may be linked to the N-terminus, the C-terminus or internally, via an amino acid side chain of the scaffold.
  • the hlL-2 may be linked to the C-terminus of the light chain or fragment thereof, to the C-terminus of the heavy chain or fragment thereof, to the N-terminus of the light chain or fragment thereof or to the N-terminus of the heavy chain or fragment thereof, or internally, via a amino acid side chain of heavy and/or light chain.
  • the hlL-2 may be linked to the N-terminus, the C-terminus or internally, via an amino acid side chain of the Fc protein.
  • an antibody comprising two hlL-2 moieties fused to the light chains or heavy chains, respectively, may be used.
  • the Fc molecule may be an Fc molecule having a native Fc sequence or a genetically engineered Fc molecule.
  • the hlL-2 portion of the hlL-2 variant used in the present invention may be prepared recombinantly or synthetically.
  • a nucleotide sequence and an amino acid sequence of wild type human IL-2 are known to a skilled person and are disclosed, for instance, in Genbank Entrez Reference 3558, as updated on updated on 3- Jun-2018, and UniProt Reference UniProtKB - P60568, as last modified on May 23, 2018, respectively.
  • the human IL-2 portion of the hlL-2 variant used in the present invention is preferably a mature hlL-2, lacking the signal peptide.
  • the human IL-2 portion of the hlL-2 variant used in the present invention may be glycosylated or unglycosylated.
  • a preferred human IL-2 portion of the hlL-2 variant which can be used in the present invention is commercially available, including for pharmaceutical uses, and it is authorized for use in human patients as aldesleukin.
  • Aldesleukin is a recombinant unglycosylated des-alanyl-1 , serine-125 human interleukin-2, recombinantly produced in E.coli.
  • Roncoleukin® is a recombinant human IL-2 produced in yeast.
  • the human IL-2 portion of the hlL-2 variant used in the present invention is aldesleukin, Roncoleukin®, or wildtype hlL-2.
  • Aldesleukin is the active ingredient of Proleukin®.
  • Aldesleukin is an unglycosylated variant of mature human IL-2 comprising two amino acid modifications as compared to the sequence of mature human IL-2: the deletion of the first amino acid (alanine) and the substitution of cysteine at position 125 by serine.
  • Mature hlL-2 protein has a molecular weight of about 15 KDa.
  • the following hlL-2 variants were successfully used: human IL-2 recombinantly fused to i) the C-terminus of the light chain of mAb MOPC-21 , ii) the C-terminus of the heavy chain of mAb DP47GS, iii) the C-terminus of the light chain of Mab DP47GS, iv) the N-terminus of human serum albumin (HAS), and v) the C-terminus of HSA.
  • the administration of the hlL-2 variant of the embodiments of the inventions begins within 5 days after the administration of ADAS, and wherein the hlL-2 variant is administered at least once daily or continuously, and is administered for at least 2 subsequent days.
  • the hlL-2 variant may be administered continuously or semi-continuously.
  • the hlL-2 variant is administered for at least 3 or 4 subsequent days, and/or is administered for between 2 and 21 subsequent days.
  • the at least once daily or continuous administration of the hlL-2 variant may be performed for up to 10, 15, 20 or 21 days or even longer.
  • the hlL-2 variant is administered for at least 3 or 4 subsequent days, and/or is administered for between 2 and 21 subsequent days.
  • the hlL-2 variant is administered for at least 3, 4, 5, 6, 7, 8, 9 or 10 subsequent days.
  • the hlL-2 variant is administered for between 3 and 21 , 4 and 21 , 5 and 21 , 3 and 15, 4 and 15, 5 and 15, 30 and 10, 4 and 10 or 5 and 10 subsequent days, such as for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 subsequent days.
  • administration of the hlL-2 variant of the embodiments of the inventions begins within 5 days after the administration of ADAS. It is preferred that administration of the hlL-2 variant begins at the day of the administration of ADAS or within 1 day, 2 days, 3 days or 4 days after said day. Therefore, in a yet further preferred embodiment, administration of the hlL-2 variant begins at the day of the administration of the peptide-loaded major histocompatibility complex class I (MHC I) presenting cell of step i) or within 1 day, 2 days, 3 days or 4 days after said day.
  • MHC I major histocompatibility complex class I
  • the hlL-2 variant may be administered by various routes.
  • the hlL-2 variant is administered system ically, locally, intravenously, subcutaneously, intraperitoneally, intratumo- rally, peritumorally or intradermally, more preferably by local, intravenous, subcutaneous, intraperitoneal, intratumoral, peritumoral or intradermal injection.
  • the hlL-2 variant is administered at a dose of from 10 to 800 pg hlL-2/kg BW per day.
  • the hlL-2 variant is administered at a dose of from 10, 20, 30, 40 or 50 to 100, 200, 300, 400 or 800 hlL-2 variant/kg BW per day.
  • the hlL-2 variant may be provided in a formulation as known to skilled persons, depending on the route of administration.
  • a solution or a dry, dried or lyophilized hlL-2 variant may be provided.
  • a dry, dried or lyophilized hlL-2 variant may be reconstituted prior to use.
  • the solution is preferably an aqueous solution, such as an aqueous buffered saline solution.
  • the formulation comprising the hlL-2 variant preferably contains one or more pharmaceutically acceptable excipients, such as water, buffering agents, such as monobasic and/or dibasic sodium phosphate, and/or mannitol. Pharmaceutically acceptable excipients are known to a skilled person.
  • ADAS in step i) pursuant to the embodiments of the present invention includes the administration of peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell to the patient.
  • MHC-I major histocompatibility complex class I
  • MHC-I major histocompatibility complex class I
  • this can be done in vitro by external loading with a peptide, such as SIINFEKL (SEQ ID No: 1 ) in the Examples as model epitope comprising peptide in the protein OVA, comprising the antigen of interest.
  • a peptide such as SIINFEKL (SEQ ID No: 1 ) in the Examples as model epitope comprising peptide in the protein OVA, comprising the antigen of interest.
  • the cells are incubated with the peptides in a suitable fluid such an aqueous solution or medium for a certain time period, such as for 10 minutes to 24 hours or 48 hours, in particular for 20 minutes to 12 hours.
  • peptides presented in the context of MHC-I typically have a length 8, 9, 10 or 11 amino acids, i.e. this length is required for binding of the peptides in the context of MHC-I, the peptides used for external loading preferably have this length.
  • the peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell of step i) in the embodiments of the present invention is obtained by incubating at least one major histocompatibility complex class I (MHC-I) presenting cell with said peptide(s) in vitro.
  • the peptide sequence may be chosen by methods known in the art.
  • Externally loading cells in vitro can be performed by methods known in the art, e.g. by providing an aqueous solution of the peptides, adding the solution to the cells, which are preferably in a buffered solution or medium, incubating the cells with the peptides as to achieve a high saturation of the MHC-I with the respective peptide, and optionally washing the cells, e.g. with an aqueous solution.
  • cells are loaded in vitro with one (1 ) defined peptide which has a sequence which is a subsequence of the antigen-comprising protein and which comprises the antigen or epitope.
  • an aqueous solution comprising one such peptide may be added in vitro to a cell population, which is preferably a cell population obtained from the patient.
  • no cell lysates are used for loading, as such cell lysates are structurally undefined and contain a huge amount of different chemical compounds, including a huge amount of different proteins.
  • MHC-I major histocompatibility complex class I
  • the cells are obtained from the patient.
  • Such mixture of cells may be obtained by incubating a suitable cell population, like a PBMC sample, with a mixture of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different peptides, thereby obtaining cells loaded with different peptides in the context of MHC-I.
  • a suitable cell population like a PBMC sample
  • separate cell populations for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cell populations
  • may be incubated in vitro with different peptides for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different peptides.
  • MHC-I major histocompatibility complex class I
  • the different peptide loaded cell major histocompatibility complex class I (MHC-I) presenting cell populations may be administered separately, or a mixture of the different peptide loaded-cell major histocompatibility complex class I (MHC-I) presenting cell populations may be prepared, which may then be administered to the patient.
  • peptides may be derived from the same or a different antigen-comprising protein.
  • 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different peptides derived from one tumor antigen may be used. This means that the sequence of each peptide is a subsequence of the tumor antigen.
  • the sequences of such different peptides may be overlapping or non-overlapping.
  • 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different peptides derived from 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different tumor antigen may be used.
  • the different tumor antigens are related to the same or different tumor, preferably to the same tumor.
  • 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different peptides derived from one infectious pathogen antigen may be used.
  • the sequence of each peptide is a subsequence of the antigen of the pathogen.
  • the sequences of such different peptides may be overlapping or non-overlapping.
  • 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different peptides derived from 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different pathogen antigens may be used.
  • the different pathogen antigens are related to the same or different pathogen, preferably to the same pathogen.
  • the tumor antigen is a neoantigen.
  • a peptide sequence of a length of 8, 9, 10, or 11 amino acids is chosen, as peptides presented by MHC-I are typically of this length.
  • HLA alleles are extremely polymorphic. Therefore, the peptide loaded is preferably a peptide presented by a frequent HLA allele. Therefore, in humans, a peptide presented by the most frequent allele HLA-A2 is particularly preferred. Alternatively, peptides presented by HLA-A1 , -A3, -B7, -B35, -C07, -C04 which are the alleles relevant for individuals of Caucasian origin may be used. HLA-A24, - A11 may be used for Asian individuals and HLA-A2, -C04, -C07 for African individuals.
  • tumor-related peptides are described in Lucasr et al. , 2010 and references therein. Most of them are HLA-A2 restricted, for example peptides derived from Melan-A/MART-1 , one of the gp100 epitopes and tyrosinase for the melanocyte differentiation antigens; prostate surface antigen and PSAP for prostate; carcinoembryonic antigen and MUC-1 for mucosal tumors; HER-2/neu for breast carcinoma; G250 for renal cell carcinoma; the PR1 shared by two myeloid leukemia associated antigens, PR3 and neutrophil elastase which are normally expressed in granulocytes and overexpressed in myeloid leukemia cells; the shared tumor-specific antigens MAGE-A and NY-ESO-1 for various tumor types; and the overexpressed proteins survivin and telomerase. Suitable Influenza A-derived peptides are described in Wu et al., 2011.
  • the peptide derived from the antigen comprising protein is a peptide presented by a MHC-I, preferably by allele HLA- A2, HLA-A1 , HLA-A3, HLA-B7, HLA-B35, HLA-A24, or HLA-A30, more preferably by allele HLA-A2.
  • Methods for identifying such peptides are described and summarized in Wu et al., 2011. For example, the systematic identification approach of Wu et al. (supra) may be used, or suitable algorithms described therein.
  • the peptide derived from the antigen-comprising protein has a length of 8, 9, 10, or 11 amino acids and/or is a peptide presented by a MHC-I, preferably by allele HLA-A2, HLA-A1 , HLA-A3, HLA-B7, HLA-B35, HLA-A24, or HLA-A30, more preferably by allele HLA-A2.
  • the peptide loaded onto the major histocompatibility complex class I (MHC-I) presenting cell comprises the antigen or epitope of interest in order to elicit the desired response.
  • the at least one peptide comprises the antigen or epitope.
  • the method of amplifying a cellular cytotoxic immune response is for prophylactically treating or treating a tumor and/or an infection, and/or wherein the patient is immunocompromised or immunosuppressed.
  • the extension and/or amplification of a cellular cytotoxic immune response is in particular for prophylactically treating or treating a tumor.
  • the patient may be suffering from tumor, or, in case of prophylactic treatment, is not suffering from a tumor, but is to be protected from a respective tumor disease. Therefore, the prophylactic treatment preferably relates to an immunization against said tumor.
  • the tumor is selected from virally induced cancer, in particular hepatitis B- or hepatitis C-induced hepatocellular carcinoma, a human papillomavirus-induced cancer, e.g. cervical, vaginal, vulvar, oropharyngeal cancer, an Epstein-Barr virus induced cancer, Kaposi sarcoma, and adult T-cell leukemia (HTLV1 ).
  • virally induced cancer in particular hepatitis B- or hepatitis C-induced hepatocellular carcinoma
  • a human papillomavirus-induced cancer e.g. cervical, vaginal, vulvar, oropharyngeal cancer
  • an Epstein-Barr virus induced cancer e.g. cervical, vaginal, vulvar, oropharyngeal cancer
  • Kaposi sarcoma e.g., Kaposi sarcoma
  • HTLV1 adult T-cell leukemia
  • the antigen-comprising protein is preferably a protein of the virus.
  • the tumor is a leukemia, in particular selected from AML, CML, CMML, and MDS.
  • the tumor is a solid cancer which expresses one or more cancer-specific antigens, such as breast cancer, prostate cancer, lung cancers, including lung squamous cell cancer and non-small cell lung cancer, skin cancers, such as melanoma, bladder cancer, esophageal adenocarcinoma and squamous cell cancer, colorectal cancer, intestinal adenocarcinoma, kidney cancers, including renal cell carcinoma, ovarian cancers, neuroblastoma, glioma, multiple myeloma, pancreatic cancer, and sarcoma.
  • cancer-specific antigens such as breast cancer, prostate cancer, lung cancers, including lung squamous cell cancer and non-small cell lung cancer, skin cancers, such as melanoma, bladder cancer, esophageal adenocarcinoma and squamous cell cancer, colorectal cancer, intestinal adenocarcinoma, kidney cancers, including renal cell carcinoma, ovarian
  • the use for the prophylactic treatment of a tumor, in particular immunization against a tumor, of a human is particularly preferred.
  • the extension and/or amplification of a cellular cytotoxic immune response is in particular for prophylactically treating or treating an infection.
  • the patient may be suffering from an infection, or, in case of prophylactic treatment, is not suffering from an infection, but is to be protected from a respective infection. Therefore, the prophylactic treatment preferably relates to an immunization against said infection.
  • the infection is an infection by a pathogen, in particular by an infectious pathogen selected from bacteria, viruses, parasites and fungi.
  • an infectious pathogen is selected from malaria, tuberculosis, leishmania and a virus, in particular a virus selected from an orthomyxovirus, influenza virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, a lentivirus, in particular Hl-Virus, cytomegalovirus, a herpes virus, a papillomavirus, a bunyavirus, a calicivirus, a filovirus, a flavivirus, and a respiratory virus, more preferably the infectious pathogen is a virus selected from a hepatitis C virus, hepatitic B virus, a papillomavirus, a paramyxovirus, and a respiratory virus.
  • the antigen-comprising protein is preferably a protein of the virus.
  • Suitable antigen-comprising proteins are known to the skilled person. The choice of an antigen-comprising protein depends on the medical indication to be considered. For the prophylactic treatment or treatment of a non-virally induced tumor, the antigen-comprising protein is a suitable tumor antigen. For the prophylactic treatment or treatment of an infection by a pathogen, the antigen comprising protein is a suitable antigen of the pathogen.
  • suitable antigen-comprising proteins and peptides comprising antigens in the context of tumor diseases are known.
  • suitable peptides as vaccines are described, as e.g. summarized in Aranda et al. , 2013 for solid neoplasms, including glioma, lung carcinoma, sarcoma, melanoma, esophageal squamous cell carcinoma, gastric cancer, hepatocellular carcinoma, pancreatic cancer, colorectal carcinoma, renal cell carcinoma, prostate cancer, ovarian carcinoma, gynecologic malignancies, and various other tumors.
  • the antigen-comprising proteins specifically targeted in these clinical trials encompass cancer-testis antigens such as NY-ESO-1 , TTK protein kinase (also known as MOS), lymphocyte antigen 6 complex, locus K (LY6K, best known as URLC10), insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3, best known as IMP3), ring finger protein (RNF43), and translocase of outer mitochondrial membrane 34 (TOMM34); carcinoembryonic antigens like glypican-3; differentiation antigens such as melan-A (MLANA) and premelanosome protein (PMEL, best known as gp100); tumor-restricted antigens, such as the SYT-SSX fusion (which is selectively expressed by synovial sarcomas as a result of a t(X; 18)(p11 ;q11 ) chromosomal translocation); as well as so-called“shared tumor- associated antigens” (
  • Suitable antigen-comprised protein involved in tumor diseases are known in the art.
  • ideal tumor antigens are those known as shared tumor-specific antigens because they are selectively expressed in tumor cells of various histotypes, and not in MFIC-expressing normal tissues.
  • Examples of such type of antigens are the MAGE-A or NY-ESO-1 antigens.
  • the various members in this category of antigens are expressed at variable proportions depending on the tumor type and the disease stage.
  • vaccines based on these antigens require selection of patients bearing tumors that express the target antigen.
  • Another category of tumor antigens that are deemed valuable for vaccine development are those derived from oncogenic proteins which are overexpressed in tumors. Bona fide non-self tumor antigens are derived from two major sources: viral antigens in the case of tumors of oncogenic viral origin such as cervical carcinomas caused by FIPV infection, and somatic mutations.
  • antigen-comprised proteins of a pathogen may be used, in particular a pathogen selected from malaria, tuberculosis, leishmania or a virus, in particular a virus selected from an orthomyxovirus, influenza virus, hepatitis A virus, hepatitis B virus, chronic hepatitis C virus, a lentivirus, in particular H l-Virus, cytomegalovirus, a herpes virus, a papillomavirus, a bunyavirus, a calicivirus, a filovirus, a flavivirus, or a respiratory virus.
  • antigen-comprising protein is a protein of a virus selected from a hepatitis C virus, a papillomavirus, a paramyxovirus, or a respiratory virus
  • Viruses in particular RNA viruses, typically exhibit a high mutation rate.
  • Flowever there are typically conserved regions found in particular in genomic segments encoding non-structural and/or internal proteins, such regions encoding a viral polymerase or a nucleoprotein. Such regions are unsuitable for classical vaccination technologies, as such conserved proteins are not exposed on the viral coat surface.
  • antigen-comprised proteins and/or antigen comprised in the protein may be used in the medicament for use according to the invention.
  • the antigen-comprising protein and/or antigen comprised in the protein of a virus is conserved.
  • the antigen-comprising protein is a non- structural protein and/or the antigen is comprised in a non-structural and/or internal protein.
  • the antigen-comprising protein is a protein of an RNA virus.
  • A“non-structural” or“internal” protein is a protein which is not part of the coat or envelope of a virus particle.
  • the antigen is comprised in the protein in step i) of the embodiments of the invention is immunodominant.
  • Immunodominant means that, although many pMHC complexes are available for a certain pathogen, T-cell responses are reproducibly focused on one or few key antigens, and such the antigen is one such key antigens.
  • Suitable antigen-comprising proteins and antigens of influenza A virus are for example described in Wu et al. , 2011 , and encompass NP (nucleoprotein), basic polymerase 1 and M1.
  • the present method is in particular beneficial for prophylactically treating, such as immunizing, or treating a tumor and/or an infection, wherein the patient is immunocompromised or immunosuppressed.
  • Patients may be immunocompromised or immunosuppressed due to transplantation, such as bone marrow transplantation, a chemotherapy, such as cancer chemotherapy, glucocorticoid therapy, leukemia, AIDS, a glucocorticoid therapy, or an immunosuppressive therapy, for example for treating an autoimmune disease.
  • a chemotherapy such as cancer chemotherapy, glucocorticoid therapy, leukemia, AIDS, a glucocorticoid therapy, or an immunosuppressive therapy, for example for treating an autoimmune disease.
  • an antigen-comprising protein may contain more than one antigen or epitope
  • the cells may be loaded with two or more different peptides, which may comprise different antigens or epitopes.
  • the two or more peptides may contain the same antigen or epitope, but may have a different length.
  • the loading can be performed in vitro by“internal” loading of the cells with antigen in a variety of ways, such as, but not limited to electroporation, transfection, or infection in vitro prior to (re-)administration into the primed host together with a Th1 adjuvant which is a danger signal (Example 7 of WO 2015/140172).
  • This procedure results in a strong amplification and expansion of the primed CD8 + T cells and triggers their further differentiation to cytotoxic cell, as shown in the Examples.
  • the unprocessed antigen comprising protein or a fragment thereof comprising the antigen(s) or epitope(s) is introduced into the MHC-I bearing cells, whereupon it is enzymatically broken down (“processed”) into a plurality of different peptides, which are then presented on the surface in the context of MHC-I (and MHC-II).
  • the transport of the unprocessed antigen-comprising protein or a fragment thereof comprising the antigen into the cell can be achieved with a variety of physical or chemical methods which are known to a skilled person, such as, but not limited to, electroporation, forced endocytosis, injection, and cell-penetrating peptides.
  • nucleic acids such as DNA or RNA
  • the introduction of nucleic acid into the cells can be by any chemical, physical, or biological means, such as, but not limited to electroporation, injection, transfection, or infection with organisms recombinantly modified to carry the nucleic acid sequence coding for the antigen comprising protein or a fragment thereof comprising the antigen.
  • suitable RNA constructs or RNA-based expression systems preferably further comprise elements which allow for translation and therefore expression of a protein in the cell of interest.
  • suitable DNA constructs or DNA-based expression systems preferably further comprise elements which allow for transcription and translation and therefore expression of a protein in the cell of interest.
  • such systems further comprise suitable elements which allow for replication of the DNA or RNA construct, or DNA- or RNA-based expression system, respectively.
  • viral systems and non-viral expression systems, which are capable of expressing a protein of interest (in this case the antigen-comprising protein or a fragment thereof comprising the antigen) in the major histocompatibility complex class I (MFIC-I) presenting cell, are suitable for this purpose.
  • a protein of interest in this case the antigen-comprising protein or a fragment thereof comprising the antigen
  • MFIC-I major histocompatibility complex class I
  • the peptide-loaded major histocompatibility complex class I (MFIC-I) presenting cell of step i) is obtained by:
  • MFIC-I major histocompatibility complex class I
  • MFIC-I major histocompatibility complex class I
  • MFIC-I major histocompatibility complex class I
  • MHC-I major histocompatibility complex class I
  • MHC-I major histocompatibility complex class I
  • MHC-I major histocompatibility complex class I
  • CPP cell-penetrating peptide
  • a compound comprising a cell-penetrating peptide (CPP) and the antigen comprising protein or a fragment thereof comprising the antigen is understood as a compound wherein a cell-penetrating peptide (CPP) is chemically linked the antigen-comprising protein or a fragment thereof comprising the antigen, optionally by a suitable linker.
  • linker may for example be a peptide linker, for example a peptide linker having a length of 1 to 50, preferably 1 to 20, more preferably 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, however, also other linkers can be used.
  • the compound is a fusion protein comprising a cell- penetrating peptide (CPP) and the antigen-comprising protein or a fragment thereof comprising the antigen, which fusion protein may optionally contain a suitable peptide linker.
  • the compound is a fusion protein consisting of a cell-penetrating peptide (CPP) and the antigen-comprising protein or a fragment thereof comprising the antigen, wherein the cell-penetrating peptide (CPP) and the antigen-comprising protein or a fragment thereof comprising the antigen are linked via a peptide bond.
  • the cell- penetrating peptide (CPP) is located N-terminally or C-terminally to the antigen comprising protein or a fragment thereof comprising the antigen in the compound.
  • the peptide-loaded major histocompatibility complex class I (MHC I) presenting cell is not cell derived from a cell line, or a cell obtained from a mammal which was cultivated in vitro for a longer time period, such as more than 3 weeks, as a longer in vitro cultivation of cells results in phenotypic changes of such cells. Accordingly, it is preferred to use primary cells, i.e. cells which are obtained from mammal and which are either not cultivated in vitro or are cultivated in vitro for up to 3 weeks, 20 days, 14 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days or 3 days.
  • primary cells i.e. cells which are obtained from mammal and which are either not cultivated in vitro or are cultivated in vitro for up to 3 weeks, 20 days, 14 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days or 3 days.
  • the peptide- loaded major histocompatibility complex class I (MHC I) presenting cell is a primary cell.
  • primary cells such as PBMC, T cells, B cells, or monocytes, as described herein, for ADAS
  • primary cells expanded to high cell numbers in vitro for ADAS These expanded cells would be either externally loaded with peptides, or internally with proteins; alternatively they would be exposed to vectors coding for peptides or proteins, as described in detail herein.
  • PBMC small PBMC sample
  • T cells or B cells from the sample by in vitro culture for up to 3 weeks, such as up to 14 days. This procedure is possible with or without prior separation of T cells or B cells.
  • the in vitro expansion process is technically feasible using GMP- compatible closed systems and employing T cell- or B-cell activating systems.
  • the T cell receptor-complex is triggered with suitable agents, e.g. antibodies, and expansion of the T cells is ensured by adding (a) growth factor(s), in particular a mixture of growth factors, typically including IL-2.
  • suitable agents e.g. antibodies
  • expansion of the T cells is ensured by adding (a) growth factor(s), in particular a mixture of growth factors, typically including IL-2.
  • growth factor(s) in particular a mixture of growth factors, typically including IL-2.
  • B cells the B- cell receptor-complex is triggered with suitable agents, followed by addition of appropriate growth factor(s).
  • the cells After expansion of T cells or B cells to high cell numbers, such as 1x10 9 to 10x10 1 °, the cells are externally loaded with peptide(s) or internally loaded with long peptides or proteins, or expression systems for long peptides or proteins, for a short period of time, such as up to 2 days, as described in detail herein and used for the ADAS procedure together with an Th1 adjuvant which is a danger signal.
  • the peptide-loaded major histocompatibility complex class I (MHC I) presenting cell is a cell selected from a monocyte, a T cell and a B cell.
  • monocytes, T cells, B cells or mixtures thereof may be loaded with peptide.
  • PBMC samples consist of T cells, B cells, NK cells, and monocytes. There is a tiny population 0.5 to 1 % of DC in peripheral blood and they are not regarded as PBMC cells.
  • PBMC samples are used as a sample for ADAS, the few contaminating DC are functionally irrelevant compared to the vast majority 99-99.5% of MHC-l-presenting cells in a PBMC sample.
  • a sample of PBMC is regarded as a mixture of T cells, B cells, NK cells, and monocytes only.
  • monocytes, T cells, B cells may be loaded by using a PBMC sample directly, or the cell types or mixtures thereof may be loaded after purification from a suitable source, such as a PBMC sample.
  • the cell is a PBMC cell or a mixture of different PBMC cells.
  • the peptide-loaded major histocompatibility complex class I (MFIC I) cell may be an allogeneic cell or an autologous cell.
  • the peptide-loaded major histocompatibility complex class I (MFIC I) presenting cell is an autologous cell.
  • the peptide-loaded major histocompatibility complex class I (MFIC I) presenting cell is not a tumor cell or hyperproliferative cell. Such cells are preferably not used in order to avoid any unwanted hyperproliferation in the patient’s body.
  • the peptide-loaded major histocompatibility complex class I (MFIC I) presenting cell is not a dendritic cell (DC).
  • the peptide-loaded major histocompatibility complex class I (MHC I) presenting cell is administered system ically.
  • the peptide-loaded major histocompatibility complex class I (MHC I) presenting cell is administered intravenously, such as by intravenous injection or infusion.
  • the peptide-loaded major histocompatibility complex class I (MHC I) presenting cell is administered at a dose of between 1x10 8 and 100x10 9 peptide-loaded major histocompatibility complex class I (MHC-I) presenting cells.
  • More preferred dose ranges are a dose of between 5x10 8 and 50x10 9 , 5x10 8 and 20x10 9 5x10 8 and 10x10 9 , 5x10 8 and 5x10 9 , 5x10 8 and 20x10 8 , between 5x10 8 and 10x10 8 , between 1x10 9 and 100x10 9 , 1x10 9 and 50x10 9 1x10 9 and 20x10 9 1x10 9 and 10x10 9 , 1x10 9 and 5x10 9 , 1x10 9 and 2x10 9 and between 1x10 8 and 2x10 9 peptide-loaded major histocompatibility complex class I (MHC-I) presenting cells.
  • MHC-I major histocompatibility complex class I
  • MHC I major histocompatibility complex class I
  • PBMC sample which comprises the cell types T cells, B cells and monocytes.
  • a population of peptide-loaded major histocompatibility complex class I (MHC I) presenting cells is administered in step i).
  • the population of peptide-loaded major histocompatibility complex class I (MHC I) presenting cells is a PBMC sample.
  • the population of peptide-loaded major histocompatibility complex class I (MHC I) presenting cells comprises T cells and/or B cells.
  • Th1 adjuvants which are danger signals make use of Th1 adjuvants which are danger signals.
  • Th1 adjuvants which are a danger signal are selected independently from each other and may be the same or different.
  • PRR Pattern Recognition Receptors
  • APC which sense Pathogen-Associated Molecular Patterns (PAMPs), which are structural components specific to microbes.
  • PRR toll-like receptors (TLR), retinoic acid-inducible gene I (RIG-I), and Melanoma Differentiation-Associated protein 5 (MDA5), NOD-like receptors, and C type lectins.
  • TLR toll-like receptors
  • RIG-I retinoic acid-inducible gene I
  • MDA5 Melanoma Differentiation-Associated protein 5
  • NOD-like receptors and C type lectins.
  • A“Th1 adjuvant which is a danger signal” induces and supports a Th1/Tc1 cytotoxic response by activating one of the PRR responsible for triggering a Th1/Tc1 immune response, including secretion of IFN-y by T cells and induction of T cell cytotoxicity.
  • PRR responsible for triggering a Th1/Tc1 immune response, including secretion of IFN-y by T cells and induction of T cell cytotoxicity.
  • Various PRR trigger a rather uniform Th1/Tc1 immune response directed at elimination of cells harboring intracellular pathogens.
  • A“Th1 adjuvant which is a danger signal” is understood to be selected from a Pathogen-Associated Molecular Pattern (PAMP, e.g. LPS), a derivative of a PAMP (e.g. MPL and GLA as derivatives of LPS), or an artificial compound, such as a peptide, protein, lipid, lipoprotein, carbohydrate, nucleic acid or small molecule, mimicking a PAMP and activating one or several Pattern Recognition Receptors (PRR).
  • PAMP Pathogen-Associated Molecular Pattern
  • MPL and GLA as derivatives of LPS
  • an artificial compound such as a peptide, protein, lipid, lipoprotein, carbohydrate, nucleic acid or small molecule, mimicking a PAMP and activating one or several Pattern Recognition Receptors (PRR).
  • PRR Pattern Recognition Receptors
  • Examples of such artificial compounds are poly (l:C), mimicking double- stranded viral RNA and activating TLR3, or CpG mimicking unmethylated
  • Table 1 lists some known Pattern Recognition Receptors (PRR) inducing a Th1/Tc1 immune response and the corresponding natural PAMP or danger signal adjuvant inducing and supporting a Th1/Tc1 cytotoxic response (Coffman et al., 2010, Wang et al., 2013). Of note, depending on the conditions, TLR2 and TLR5 may alternatively induce a non-cytotoxic Th2 T cell response.
  • PRR Pattern Recognition Receptors
  • Th1 adjuvants which are a danger signal listed in Table 1 are preferred as “Th1 adjuvant which is a danger signal” of the present invention.
  • Table 1 The Th1 adjuvant which is a danger signal can be administered temporally and/or spatially separate or together with ADAS. When administered temporally and spatially separate, the Th1 adjuvant which is a danger signal is preferably administered within 24 h after administration of ADAS. When administered temporally together with ADAS, the Th1 adjuvant which is a danger signal may be covalently or non-covalently attached to the cells used for ADAS or may be not attached to the cells, and may be in the same composition as the cells or in a separate composition, such as a separate vial or container.
  • DNA- or RNA-vaccines contain nucleic acid sequences which trigger PRR. Therefore, these vaccines do not require a separate administration of a Th1 adjuvant which is a danger signal; these vaccines are called“self-adjuvanted” vaccines.
  • peptide or protein vaccines it is possible to covalently bind them to another chemical moiety, by fusing or chemically attaching a protein or peptide sequence, such as flagellin, microtubule elongation factors and derived peptides/proteins, Hsp70359-610 (Wang et al., 2002) or HMGB1 -derived peptides (https://techtransfer.universityofcalifornia.edu/NCD/21443.html), which triggers at least one appropriate PRR and thus induce a Th1/Tc1 immune reaction.
  • a protein or peptide sequence such as flagellin, microtubule elongation factors and derived peptides/proteins, Hsp70359-610 (Wang et al., 2002) or HMGB1 -derived peptides (https://techtransfer.universityofcalifornia.edu/NCD/21443.html
  • the Th1 adjuvant which is a danger signal is recognized by a Pattern Recognition Receptor (PRR).
  • PRR Pattern Recognition Receptor
  • the Th1 adjuvant which is a danger signal is or mimicks a pathogen-associated molecular pattern (PAMP).
  • cytokines such as IL-2
  • IL-2 are wrongly designated as being“adjuvants” in the context of T cell responses.
  • Such wrong designation is both attributable to the incorrect use of the term“adjuvant”, which is generally accepted to refer to an agent which modifies the effect of other agents while having few if any direct effects when given by itself.
  • adjuvants are drugs that have few or no pharmacological effects by themselves, but may increase the efficacy or potency of other drugs when given at the same time.
  • an adjuvant is an agent which, while not having any specific antigenic effect in itself, may stimulate the immune system, increasing the response to a vaccine.
  • Cytokines, such as IL-2 are known to have numerous effects on their own, and do not represent an“adjuvant” according to the present invention. In any event, cytokines, such as IL-2 are not“Th1 adjuvants which are a danger signal” according to the present invention.
  • the Th1 adjuvant which is a danger signal is not a cytokine.
  • the Th1 adjuvant which is a danger signal is not IL-2.
  • the Th1 adjuvant which is a danger signal is administered systemically, locally, intravenously, subcu taneously, intraperitoneally, intratumorally or intradermally and/or by injection.
  • Th1 adjuvant which is a danger signal for use of the present invention
  • PRR Pattern Recognition Receptor
  • the pathogen-associated molecular pattern is selected from cGAMP, c-di-GMP, c-di-AMP, dsRNA, MALP-2, a peptidoglycan, a lipopeptide, lipotechoic acid, lipopolysaccharide (LPS), RSV fusion protein, Flagellin, ssRNA, an imidazoquinolone, and a CpG poly- or oligodeoxynucleotide, and/or
  • the Th1 adjuvant which is a danger signal is selected from a STING agonist, preferably a STING activating cyclic dinucleotide or a small molecule, a RIG-I agonist, preferably selected from poly (I: C), polylCLC, poly l:C12U, polyl:C12C, an RNA-based RIG-I agonist, and a small molecule RIG-I agonist, an MD5 agonist preferably selected from poly (I: C), polylCLC, poly l:C12U, polyl:C12C, an RNA-based MD5 agonist, and a small molecule MD5 agonist, a TLR2 agonist, preferably selected from Pam3CSK4, Pam2Cys, Pam3Cys, lipoproteins, porins, toxins, and small molecule TLR2 agonists, a TLR3 agonist, preferably selected from poly (I: C), polylCLC, ARNAX, poly l:C12U, poly
  • the patient is a mammal, preferably a primate, more preferably a primate selected from Hominidae, even more preferably a human.
  • Preferred primates are humans.
  • the patient is a human.
  • the invention relates in one embodiment to a medicament for use in a method of amplifying a cellular cytotoxic immune response against an antigen-comprising protein in a patient, the method comprising the steps of:
  • the peptide is derived from the antigen-comprising protein, thereby re-activating the activated T cell, wherein the peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell is administered only once and in a time frame of from 4 days to 29 days after the T cells were activated against an antigen, and
  • MHC-I major histocompatibility complex class I
  • hlL-2 variant administered at least once daily or continuously, and is administered for at least 2 subsequent days.
  • naive CD8 + T cells can be activated by i) viruses (such as e.g. hepatitis B and C viruses, cytomegalovirus), ii) bacteria, iii) fungi, iv) parasites, or by v) tumor antigens, including neoantigens.
  • viruses such as e.g. hepatitis B and C viruses, cytomegalovirus
  • bacteria such as e.g. hepatitis B and C viruses, cytomegalovirus
  • fungi e.g. fungi
  • iv tumor antigens
  • tumor antigens including neoantigens.
  • CD8 + T cells in a host can be activated by therapeutic vaccines based on microbes, such as live, attenuated (e.g. MVA-vaccine), or killed, recombinant bacteria or viruses, and vectors derived from viruses.
  • T cells can be activated by peptides, proteins, or nucleic acids, which may be DNA and RNA, coding for a peptide or protein.
  • the vaccine is taken up by professional APC and the (encoded) antigen processed to peptides, which are presented on the surface in the context of MHC-I and MHC-II.
  • CD8 + T cells recognize a presented peptides in the context of MHC-I and become fully activated, i.e. cytotoxic against an antigen if the therapeutic vaccine contains a Pathogen-Associated Molecular Pattern (PAMP) or if a Th1 adjuvant which is a danger signal is included in the immunization procedure.
  • PAMP Pathogen-Associated Molecular Pattern
  • a plurality of alternative options available for activating T cells against an antigen may be used, as described previously.
  • a targeted delivery system may be used, as described previously.
  • alternative for targeted and non-targeted approaches are known in the art. The alternative approaches are known to result in varying numbers of T cells activated against the antigen.
  • a microbe bacteria or virus
  • vectored vaccines which include viral vectors expressing heterologous antigens
  • an antigenic protein or peptide can be injected into the host
  • the protein or peptide can also be fused to a cell- penetrating peptide Brooks et al., 2010
  • nucleic acids such as RNA or DNA, encoding a peptide or protein can be administered by transcutaneous electroporation; vi) the protein or peptide or the encoding nucleic acid can be re
  • nanoparticles which are understood as nano-scale size materials made of polymers, peptides, proteins or lipids, in particular PLGA, liposomes, virosomes, virus-like particles (Trovato et al., 2015).
  • Modern known delivery systems are designed to directly target the antigen to an APC (macrophages, B cells, DC, e.g. WO 2015/140172):
  • Monoclonal antibodies (mAbs) and mAb-derived compounds can be used for targeting peptides/proteins or nucleic acids to surface structures on APC, in particular on DC, such as CD141.
  • DC such as CD141.
  • the mAbs preferentially recognize DC- specific surface receptors such as XCR1 , nectin-like molecule 2, and c-type lectin- 9A (CLEC9A).
  • Alternative targeting systems are natural or synthetic ligands, including peptides, proteins, carbohydrates, aptamers, binding to receptors expressed on APC, e.g. XCL1 , as described in detail in WO 2009/065561.
  • Antibody-based or ligand-based targeting systems can also be used to functionalize a variety of carriers, so that the carrier-bound antigenic protein/peptide or the encoding nucleic acid are targeted to APC.
  • Preferential targeting to APC can also be achieved by a particular formulation of the carrier, such as a liposome (Schwendener et al. , 2014).
  • Preferred delivery systems are described below in detail.
  • a cytotoxic CD8 + T cell response will result.
  • the mode of antigen delivery only differs in its potency for inducing a Th1/Tc1 CD8 + T cell response.
  • the Th1 danger signal can be omitted in case of self-adjuvanted vaccines.
  • the T cells are activated against the antigen by a method of (i) or (ii):
  • the protein of (b) is internalized and processed in the antigen-presenting cell and the antigen comprised in the protein is presented on the surface of the antigen- presenting cell, thereby activating a T cell in the patient, and
  • Th1 adjuvant which is a danger signal administering a Th1 adjuvant which is a danger signal, wherein the Th1 adjuvant which is a danger signal is optionally covalently or non-covalently bound to the delivery system;
  • an antigen-comprising protein or a fragment thereof comprising the antigen or a compound comprising the antigen-comprising protein or fragment thereof, wherein the antigen-comprising protein or a fragment is optionally in a composition comprising one or more carrier(s) and/or auxiliary agent(s), or
  • nucleic acid comprising a nucleic acid sequence encoding an antigen comprising protein or a fragment thereof comprising the antigen, wherein the nucleic acid is capable of expressing the antigen-comprising protein or a fragment thereof comprising the antigen in said patient, or a compound comprising said nucleic acid, or a host cell, virus or viral system comprising said nucleic acid, wherein the nucleic acid or compound is optionally in a composition comprising one or more carrier(s) and/or auxiliary agent(s), thereby activating at least one T cell,
  • Th1 -adjuvant which is a danger signal
  • Th1 adjuvant which is a danger signal is optionally covalently or non- covalently bound to i) the antigen-comprising protein or fragment thereof, or molecule comprising the antigen-comprising protein or fragment thereof, ii) the nucleic acid, or molecule comprising the nucleic acid, or, iiii) host cell, virus or viral system comprising the nucleic acid.
  • the nucleic acid is DNA or RNA.
  • the nucleic acid is an expression system.
  • Such expression system allows for expression of the antigen in a cell of the patient.
  • the host cell, virus or viral system comprising said nucleic acid which is capable of expressing the antigen comprising protein or a fragment thereof comprising the antigen in said patient is selected from an attenuated virus, an attenuated bacterium, a non-replicating viral system, a targeted viral vaccine system or non-targeted viral vaccine system.
  • the targeted viral vaccine system displays on its surface a moiety specifically recognizing a cell surface marker of the Antigen- Presenting Cell (APC).
  • APC Antigen- Presenting Cell
  • the compound comprising the antigen comprising protein or fragment thereof is a fusion protein comprising a cell penetrating peptide (CPP), or is an antigen-comprising protein or fragment thereof covalently linked to a moiety selected from a nucleic acid or peptide and/or to a moiety mimicking a pathogen-associated molecular pattern (PAMP).
  • CPP cell penetrating peptide
  • PAMP pathogen-associated molecular pattern
  • A“cell-penetrating peptide” also known as protein transduction domain (PTD), membrane translocating sequence (MTS), is understood as a peptide of 3 to 40 amino acids, which is able to penetrate into almost any cell.
  • the cell-penetrating peptide is highly cationic and/or contains a high content of arginine and/or lysine amino acids.
  • a preferred cell-penetrating peptide is R9, which is a peptide consisting of 9 Arginine residues.
  • the carrier is selected from a liposome, a vesicle, a nanoparticle, and a microparticle, wherein the liposome, vesicle, nanoparticle, or microparticle is optionally targeted to an Antigen-Presenting Cell (APC), more preferably wherein the liposome, vesicle, nanoparticle, or microparticle targeted to an Antigen-Presenting Cell (APC) displays on its surface a moiety specifically recognizing a cell surface marker of the Antigen-Presenting Cell (APC).
  • APC Antigen-Presenting Cell
  • a method of alternative i) is used, i.e. a delivery system is used for activating a T cell against an antigen. Accordingly, in another preferred embodiment of the present invention, a delivery system of above alternative i) is administered, wherein the antigen-presenting cell is selected from a dendritic cell, a macrophage and a B cell.
  • the antigen-presenting cell is a dendritic cell
  • the receptor on the surface of a dendritic cell is a receptor on the surface of cross-presenting dendritic cells selected from chemokine (C motif) receptor 1 (XCR1 ), nectin-like molecule 2, and a c type lectin 9A (CLEC9A), or wherein the antigen-presenting cell is a XCR1 + dendritic cell, even more preferably wherein the antigen-presenting cell is a CD141 + /XCR1 + dendritic cell.
  • C motif chemokine receptor 1
  • nectin-like molecule 2 a c type lectin 9A
  • CLEC9A c type lectin 9A
  • the receptor on the surface of a dendritic cell is a receptor on the surface of cross- presenting dendritic cells.
  • Dendritic cells can present exogenous antigens via MHC class I molecules, a process known as“cross-presentation”. Therefore, in one preferred embodiment, the delivery system in a medicament for use comprises, preferably consists of, (a) a molecule binding to a receptor on the surface of a dendritic cell, and (b) an antigen-comprising protein bound to molecule of (a).
  • Cross-presenting dendritic cells are particularly suitable for presenting peptides in the context of MHC-I.
  • a cross-presenting DC is capable to take up soluble or targeted protein, process it, and present it in the context of MHC-I. All conventional DC are capable of antigen cross-presentation. Quantitatively optimal antigen cross-presentation is done by XCR1 + DC in the mouse and by XCR1 + DC within the BDCA3 + DC population in the human. Therefore, XCR1 + DC are preferred murine cross-presenting DC, and XCR1 + DC within the BDCA3 + DC population are preferred human cross-presenting DC. In a more preferred embodiment, a CD141 + /XCR1 + DC are preferred murine cross- presenting DC, and a CD141 + /XCR1 + DC within the BDCA3 + DC population are preferred human cross-presenting DC.
  • the receptor on the surface of a dendritic cell is chemokine (C motif) receptor 1 (XCR1 ), nectin-like molecule 2, a c-type lectin (CLEC) such as CLEC9A.
  • C-type lectins are Ca++-dependent glycan-binding proteins that share primary and secondary structural homology in their carbohydrate-recognition domains (CRDs). These proteins have a C-type lectin fold, which is a fold with highly variable protein sequence that is also present in many proteins that do not bind carbohydrates.
  • the sequence of the human receptor CLEC9A is for example described in Caminschi et al. , 2008, Blood 112, 3264-3273.
  • a suitable molecule binding to CLEC9A is e.g. a specific monoclonal anti-CLEC9A antibody.
  • the human receptor nectin-like molecule 2 is described in Takai et al., 2003.
  • a suitable molecule binding to nectin-like molecule 2 is e.g. a specific anti-nectin-like molecule 2 monoclonal antibody.
  • XCR1 is a chemokine receptor and is so far the only member of the "C" sub-family of chemokine receptors.
  • the natural ligand of XCR1 is XCL1 , which is also known as ATAC, lymphotactin or SCM-1.
  • An exemplary method to produce XCL1 in biologically active form is described in Example 8 of WO 2009/065561. Analogous methods may be used in order to produce other biologically active forms of XCL1 , e.g. those of other species.
  • the receptor on the surface of a dendritic cell is XCR1.
  • the amino acid sequence of human XCR1 is known (NCBI; accession NP_001019815).
  • the molecule of a) is a ligand to the receptor or an antibody or antibody fragment against the receptor.
  • the receptor is chemokine (C motif) receptor 1 (XCR1 ) and the molecule of a) is anti-XCR1 antibody or fragment thereof or chemokine (C motif) ligand 1 (XCL1 ) or a functionally active variant thereof.
  • XCL1 The amino acid sequences of XCL1 (ATAC) of several species (including human: GenBank accession P47992, entry of September 12, 2018; mouse: GenBank accession P47993; and rat: GenBank accession P51672) are known. Additionally, a specific XCLR1 agonist referred to as K4.1 HHV8 (GenBank accession AAB62672.1 ), which is a viral chemokine-like protein, is also known. Any of these naturally occurring XCR1 ligands or any other natural occurring XCR1 ligand may be used.
  • a suitable monoclonal anti-XCR1 antibody for use according to the invention is for example mAb 6F8 disclosed in WO 2009/065561 or MARX10 (Bachem et al. , 2012, Front Immunol 3, 214).
  • An anti-XCR1 antibody or functionally active fragment thereof may be used in one preferred embodiment as molecule binding to XCR1 as receptor in step i) of above alternative i) relating to a delivery system.
  • An anti-XCR1 antibody or functionally active fragment thereof is capable of binding specifically to the XCR1.
  • the functionally active fragment of the antibody is defined analogously to the functionally active fragment of XCL1 (see above), i.e.
  • the functionally active fragment (a) is characterized by being derived from any anti-XCR1 antibody by one or more amino acid deletions, such as C-terminal, N-terminal and/or internal deletions and (b) is characterized by having a biological activity similar to that displayed by the anti-XCR1 antibody from which it is derived, including the ability to binding to XCL1.
  • Naturally occurring antibodies are proteins used by the immune system to identify and neutralize foreign objects. Each naturally occurring antibody has two large heavy chains and two small light chains and can bind to a different antigen.
  • the present invention includes, for example, monoclonal and polyclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, Fab, Fab', F(ab')2', Fv, or the product of a Fab expression library.
  • the antibody or antibody component can further be modified to prolong its biological half-life or in other ways to make them more suitable for targeting.
  • Antibodies generated against XCR1 can be obtained by direct injection of XCR1 or a fragment thereof into an animal or by administering XCR1 or a fragment thereof to an animal, preferably a non-human. The antibody so obtained will then bind to XCR1.
  • any technique known in the art which provides antibodies produced by continuous cell line cultures, e.g. a hybridoma cell line, can be used.
  • the production of a suitable monoclonal antibody is also detailed in WO 2009/065561.
  • Techniques described for the production of single chain antibodies can be adapted to produce single chain antibodies to XCR1.
  • transgenic mice or other organisms such as other mammals may be used to express humanized antibodies to XCR1.
  • a delivery system is used, wherein
  • the antigen-comprising protein of (b) is in a fusion protein with the molecule of a);
  • the antigen of the antigen-comprising protein of (b) is an immunogen, a pathogen-derived antigen, or a tumor antigen.
  • the antigen-comprising protein of (b) is in a fusion protein with the molecule of a).
  • fusion proteins can be synthesized e.g. synthetically or recombinantly, preferably recombinantly.
  • Such fusion proteins enable efficient targeting to the dendritic cells.
  • fusion proteins of antibody MARX10 with a peptide comprising the antigen and XCL1 with a peptide comprising the antigen can be prepared.
  • Such delivery systems as described above are suitable delivery systems for “priming”.
  • An immunogen is an antigen that stimulates an immune response.
  • Antigens are substances recognized by specific receptors on T cells (T-cell receptor) and B cells (B-cell receptor) within the immune system and are usually proteins or polysaccharides. This includes parts (coats, capsules, cell walls, flagella, fimbrae, and toxins) of bacteria, viruses, and other microorganisms. In general, lipids and nucleic acids are antigenic only when combined with proteins and polysaccharides.
  • Non-microbial exogenous (non-self) antigens can include pollen, egg white, and proteins from transplanted tissues and organs or on the surface of transfused blood cells.
  • Antigens can be categorized as endogenous or exogenous.
  • the medicament is preferably for use in inducing a cellular cytotoxic immune response to an infection with such pathogen.
  • a pathogen-derived antigen preferably of a virus, bacterium and/or eukaryotic parasite
  • the medicament is preferably for use in inducing a cellular cytotoxic immune response to an infection with such pathogen.
  • Another preferred antigen of the present invention is an endogenous antigen. Suitable endogenous antigens are tumor antigens, in particular neoantigens.
  • the medicament is preferably for use in inducing a cellular cytotoxic immune response to such tumor.
  • Th1 adjuvant which is a danger signal is administered.
  • suitable Th1 adjuvants which are danger signals are administered as separate chemical moieties.
  • the Th1 adjuvant which is a danger signal of alternative i) of the “priming” method described above is covalently bound to the delivery system.
  • the Th1 adjuvant which is a danger signal of alternative ii) of ) of the“priming” method described above is covalently bound to i) the antigen-comprising protein or fragment thereof, or molecule comprising the antigen-comprising protein or fragment thereof, ii) the nucleic acid, or molecule comprising said nucleic acid, or, iiii) the host cell, virus or viral system comprising said nucleic acid.
  • such covalently bound Th1 adjuvant which is a danger signal is selected from a nucleic acid or peptide and/or is mimicking a pathogen-associated molecular pattern (PAMP) and/or the nucleic acid or, the host cell, virus or viral system comprising said nucleic acid itself contain a Th1 adjuvant which is a danger signal.
  • PAMP pathogen-associated molecular pattern
  • agents administered to a patient pursuant to the present invention are administered as pharmaceutical composition comprising the respective agents.
  • the pharmaceutical compositions used in the present invention comprise a therapeutically effective amount of the respective agent, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans.
  • carrier refers to a diluent, excipient, or vehicle with which the agent is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, including but not limited to peanut oil, soybean oil, mineral oil, sesame oil and the like. As explained above, preferred routes of administration apply for the respective agents used in the present invention.
  • water is a preferred carrier when the pharmaceutical composition is administered orally.
  • Saline and aqueous dextrose are preferred carriers when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions are preferably employed as liquid carriers for injectable solutions, such as for intravenous injection, intraperitoneal injection, intratumoral injection, peritumoral injection, subcutaneous injection or intradermal injection, for intravenous, intraperitoneal, intratumoral, peritumoral injection, subcutaneous or intradermal administration.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.
  • compositions will contain a therapeutically effective amount of the respective agent, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water- free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of the respective agent.
  • composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water or saline for injection can be provided so that the ingredients may be mixed prior to administration.
  • the agents of the invention can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with free carboxyl groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., those formed with free amine groups such as those derived from isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc., and those derived from sodium, potassium, ammonium, calcium, and ferric hydroxides, etc.
  • the amount of the respective agent of the invention, which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. Preferred amounts and dosages are provided for the respective agents above.
  • in vitro assays may optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose- response curves derived from in vitro or animal model test systems.
  • a therapeutic of the invention e.g., encapsulation in liposomes, microparticles, and microcapsules: use of recombinant cells capable of expressing the therapeutic, use of receptor-mediated endocytosis; construction of a therapeutic nucleic acid as part of a retroviral or other vector, etc.
  • methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • the compounds may be administered by any convenient route, for example by infusion, by bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral, rectal and intestinal mucosa, etc.), and may be administered together with other biologically active agents. Unless indicated otherwise for specific agents used in the present invention above, administration can be systemic or local.
  • compositions may be desirable to administer the pharmaceutical compositions locally to the area in need of treatment.
  • This may be achieved by, for example, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non- porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • administration can be by direct injection at the site (or former site) of a malignant tumor or neoplastic or pre-neoplastic tissue.
  • the agent can be delivered in a vesicle, in particular a liposome, such as a cationic liposome.
  • the agent can be delivered via a controlled release system.
  • a pump may be used.
  • a controlled release system can be placed in proximity of the therapeutic target, thus requiring only a fraction of the systemic dose.
  • kits or Kit-of-parts comprising
  • MHC-I major histocompatibility complex class I
  • hlL-2 variant with extended circulating half-life in vivo in a human as compared to hlL-2, wherein the hlL-2 variant is capable of binding to the high affinity IL-2 receptor chain (CD25),
  • the peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell may be comprised in a suspension, such as cells suspended in an aqueous solution and optionally further containing pharmaceutically acceptable excipients.
  • a container such as a vial, may contain the pharmaceutical composition comprising the peptide-loaded major histocompatibility complex class I (MHC-I) presenting cells.
  • the pharmaceutical composition is suitable for systemic administration, in particular for intravenous administration.
  • the pharmaceutical composition may further comprise at least one Th1 adjuvant which is a danger signal, or may not comprise Th1 adjuvant which is a danger signal.
  • the hlL-2 variant with extended circulating half-life in vivo in a human as compared to hlL-2, wherein the hlL-2 variant is capable of binding to the high affinity IL-2 receptor chain (CD25) may be comprised in a solution, such as an aqueous solution, or dried or freeze-dried, and optionally further containing pharmaceutically acceptable excipients.
  • a container, such as a vial may contain the pharmaceutical composition comprising hlL-2 variant with extended circulating half-life in vivo in a human as compared to hlL-2, wherein the hlL-2 variant is capable of binding to the high affinity IL-2 receptor chain (CD25).
  • the at least one Th1 adjuvant which is a danger signal may be comprised in a solution, such as an aqueous solution, or dried or freeze-dried, and optionally further containing pharmaceutically acceptable excipients. Therefore, the kit or kit- of-parts may further contain a pharmaceutical composition comprising at least one Th1 adjuvant which is a danger signal.
  • the pharmaceutical composition does not contain a peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell for use of the invention.
  • MHC-I major histocompatibility complex class I
  • the present invention relates to a method of amplifying a cellular cytotoxic immune response against an antigen-comprising protein in a patient, the method comprising the steps of:
  • the peptide is derived from the antigen-comprising protein, thereby re-activating the activated T cell, wherein the peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell is administered only once and in a time frame of from 4 days to 29 days after the T cells were activated against an antigen, and
  • MHC-I major histocompatibility complex class I
  • hlL-2 variant administered at least once daily or continuously, and is administered for at least 2 subsequent days.
  • the present invention relates to a method of amplifying a cellular cytotoxic immune response against an antigen-comprising protein in a patient, the method comprising the steps of:
  • step (x) administering to the patient of step (x) a peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell and a Th1 adjuvant which is a danger signal,
  • MHC-I major histocompatibility complex class I
  • the peptide is derived from the antigen-comprising protein, thereby re-activating the activated CD8 + T cell(s), wherein the peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell is administered only once and is administered in a time frame of from 4 days to 29 days after the one or more T cell(s) administered to the patient in step (x) were activated against an antigen, and
  • MHC-I major histocompatibility complex class I
  • the present invention relates to a method of amplifying a cellular cytotoxic immune response against an antigen-comprising protein in a patient, the method comprising the steps of:
  • peptide-loaded major histocompatibility complex class I MHC-I
  • Th1 adjuvant which is a danger signal
  • the peptide is derived from the antigen-comprising protein
  • T cell(s) activated against an antigen at least 30 days before, and
  • hlL-2 variant administered at least once daily or continuously, and is administered for at least 2 subsequent days.
  • Fig. 1 The combination of ADAS and IL-2ext is capable to massively expand antigen-specific CD8 + T cells, irrespective of the initial immunization mode.
  • C57BL/6 mice were immunized (“priming”) with high amounts of non-targeted OVA (200 pg) or low amounts of OVA targeted to B cells (1 D3-OVA, 2 pg) or dendritic cells (muXCL1 -OVA, 2 pg) and 10 pg poly (l:C) on day 0.
  • SIINFEKL SEQ ID No: 1
  • -peptide-loaded cells (10x10 6 ) and 50 pg poly (l:C) were applied to some animals (“priming + ADAS”).
  • IL-2ext is highly superior to wildtype IL-2 for expansion of antigen- specific CD8 + T cells after ADAS re-activation.
  • C57BL/6 mice were immunized with muXCL1 -OVA (2 pg) and 10 pg poly (I: C) on day 0 (priming).
  • SIINFEKL-loaded cells (1x10 6 ) and 50 pg poly (l:C) were applied to some animals (“priming + ADAS).
  • mice received ADAS and 3.95 pg MOPC-IL-2 (“priming + ADAS + IL-2ext”) or ADAS and equimolar standard human IL-2 (0.8 pg) (“priming + ADAS + IL-2”) or ADAS and 10-fold (8 pg) equimolar standard human IL-2 (“priming + ADAS + IL-2 (10x)”) injected i.p. on days 6, 7 and 9.
  • the proportion of SIINFEKL-specific CD8 + T cells within all CD8 + T cells was determined for all animals in the spleen on day 10.
  • IL-2cx has comparable effects and adverse effects as various preparations of IL-2 with extended half-life (IL-2ext).
  • C57BL/6 mice were immunized with muXCL1 -OVA (2 pg) and 10 pg poly (l:C) on day 0.
  • SIINFEKL-loaded cells 0.5x10 6
  • 50 pg poly I: C
  • mice were injected i.p.
  • hlL-2cx (4 pg mAb 5355 and 0.83 pg IL-2) or equimolar amounts of hlL-2 in preparations with extended half-life (IL-2ext): MOPC-IL-2 (3.95 pg), DP47GS-HC-IL-2 (3.9 pg), DP47GS-LC-IL-2 (3.9 pg), IL-2-HSA (3.82 pg), or FIAS-IL-2 (3.82 pg).
  • MOPC-IL-2 (3.95 pg)
  • DP47GS-HC-IL-2 (3.9 pg)
  • DP47GS-LC-IL-2 (3.9 pg)
  • IL-2-HSA 3.82 pg
  • FIAS-IL-2 3.82 pg
  • mice were immunized as before, with the exception that ADAS was performed on day 5 with 10x10 6 cells.
  • IL-2ext MOPC-IL-2
  • mlL-2cx MOPC-IL-2
  • IL-2ext MOPC-IL-2
  • mlL-2cx 2.3 pg murine IL-2 complexed with 10 pg mAb JES6- 5H4
  • mlL-2cx mAb JES6- 5H4
  • C57BL/6 mice were immunized with muXCL1 -OVA (2 pg) and 10 pg poly (I: C) on day 0 (priming).
  • SIINFEKL-loaded cells (1x10 6 ) and 50 pg poly (I: C) were applied i.v. (priming + ADAS).
  • Additional 3-day treatment with 3.95 pg MOPC-IL-2 i.p. (priming + ADAS + IL-2ext) began either on day 7, or days 8, 9, 10, or 11.
  • the proportion of SIINFEKL- specific CD8 + T cells in the spleen was determined one day after last application of MOPC-IL-2 in each experimental group.
  • Fig. 5 The combination of ADAS and IL-2ext re-activates and expands CD8 + T cells when applied 3 to 29 days after primary activation.
  • mice were immunized with muXCL1 -OVA (2 pg) and 10 pg poly (I: C) on day 0, peptide-loaded cells (10x10 6 ) and 50 pg poly (l:C) (priming+ADAS) were applied either (A) on days 5, 6, 7, 8, or 9, or (B) on days 3, 6, 17, or 28.
  • Some of the mice were further treated with MOPC- IL-2 (3.95 pg, i.p.) for 3 days, beginning one day after ADAS, and the levels of SIINFEKL-specific CD8 + T cells were determined in the spleen one day after the last IL-2ext application.
  • Fig. 6 The combination of ADAS and IL-2ext re-activates and expands memory T cells.
  • C57BL/6 mice were immunized with muXCL1 -OVA (2 pg) and 10 pg poly (I: C) on day 0 (priming).
  • SIINFEKEL-loaded cells (10x10 6 ) and 50 pg poly (I: C) were applied on day 5 (priming + ADAS).
  • MOPC-IL-2 3.95 pg
  • injected i.p. on days 6, 7 and 8 primary + ADAS + IL-2ext.
  • the proportion of SIINFEKL-specific CD8 + T cells was determined (A) on day 9 (initial activation phase) and day 40 (memory phase) in the blood.
  • mice All mice were re-challenged with ADAS (10x10 6 SIINFEKL-loaded cells and 50 pg poly (l:C)) on day 40 and treated with MOPC-IL-2 (3.95 pg, i.p.) on days 41 , 42, and 43, and the levels of SIINFEKL-specific CD8 + T cells were determined on day 44 in the spleen.
  • ADAS 10x10 6 SIINFEKL-loaded cells and 50 pg poly (l:C)
  • MOPC-IL-2 3.95 pg, i.p.
  • Fig. 7 The combination of ADAS and IL-2ext massively expands recently in vitro activated and adoptively transferred CD8 + T cells.
  • OT-I T cells (2x10 6 /ml) were activated with SIINFEKL-peptide (1 ng/ml final) for 3 days in vitro and then adoptively transferred into syngeneic C57BL/6 mice (0.5x10 6 cells/animal) on day 0, resulting in a frequency of 1 % of all CD8 + T cells.
  • SIINFEKL-loaded syngeneic cells (10x10 6 ) and 50 pg poly (l:C) were injected i.v. (ADAS), raising the frequency to 10%.
  • mice were further injected with MOPC-IL-2 on days 6, 7, and 8 (ADAS+IL-2ext), resulting in a frequency of 90%.
  • the proportion of OT-I CD8 + T cells in the spleen was determined for all groups of mice on day 10 using an antibody specific for Thy1 .1 expressed on OT-I T cells.
  • Some animals were treated with ADAS alone on day 28 after transfer, resulting in a frequency of around 25 % OT-I T cells.
  • Other animals further received MOPC-IL-2 on days 29, 30, and 31 (ADAS+IL-2ext), giving a frequency of 90%.
  • any Th1 adjuvant which is a danger signals by activating an appropriate PRRs in APC can be used both for the initial priming procedure and the subsequent ADAS re-activation . Therefore, the use of polyl:C here is exemplary for any Th1 adjuvant which is a danger signal which could be used instead.
  • muXCLI is the chemokine ligand for XCR1 and can be used for targeting of antigen to XCR1 + DC in the mouse (Hartung et al. , 2015).
  • MAb 1 D3 recognizes CD19 on B cells (Krop et al., 1996, obtained from ATCC), mAb MOPC-21 (Potter et al. , J Natl Cancer Inst 26 (1961 ) 1109-1137, obtained from Biolegend) does not recognize any molecule in the mouse.
  • Ovalbumin (OVA) obtained from Calbiochem, was treated to remove endotoxin.
  • SIINFEKL SEQ ID No: 1
  • SIINFEKL is the immunodominant peptide in C57BL/6 mice after immunization with OVA.
  • the antigen-binding regions of the heavy and light chains of mAb 1 D3 and MOPC-21 were identified by mass spectrometry and grafted onto the backbone of mAb DEC-205 by standard recombinant techniques, as described previously for mAb MARX10 (Hartung et al., 2015). This backbone has been modified previously to minimize binding to Fc-receptors. Recombinant mAb 1 D3 was then modified in such a way as to accommodate full-length chicken OVA as heavy chain C-terminal fusion protein, as described for mAb MARX10-OVA (Hartung et al. 2015). muXCL1 -OVA was generated as described previously (Hartung et al., 2015).
  • the IL-2 containing heavy chain and the DP47GS kappa light chain (disclosed in US 2014/0044675 A1 as“SEQ ID NO 19” and“SEQ ID No: 20”, respectively) were cloned separately into pTT5 vectors and co-expressed in HEK 293 cells.
  • human IL-2 was fused to the C-terminus of the kappa light chain of DP47GS via a (GGGGS)3 (SEQ ID No: 2)-linker.
  • the IL-2 containing kappa light chain and DP47GS heavy chain were cloned separately into pTT5 vectors and co-expressed in HEK 293 cells.
  • All antibody fusion construct were purified from HEK 293 culture supernatants using Protein A affinity chromatography.
  • human IL-2 was fused via a (GGGGS)3-linker to the N-terminus of human serum albumin, and a StrepTag sequence was added via a GTG-linker to the C-terminus.
  • human IL-2 was fused via a (GGGGS)3 (SEQ ID No: 2)- linker to the C-terminus of human serum albumin; to the C-terminus of human IL-2 a StrepTag sequence was added via a GTG-linker.
  • Both HSA constructs were cloned into pTT5 vectors and expressed in HEK 293 cells. Proteins were purified from HEK 293 culture supernatants using StrepTrap HP columns from GE Healthcare.
  • OT-I TCR-transgenic mice (Hogquistet al. , 1994) were crossed onto B6.PL mice to allow identification of adoptively transferred cells with the CD90.1 marker.
  • Complexed murine IL-2 (mll_-2cx) was obtained by incubating murine IL-2 (2.3 pg, generated and purified in the laboratory) and the anti-IL-2 mAb JES6-5H4 (10 pg, Sander et al., 1993), or human IL-2 (0.83 pg, generated and purified in the laboratory) and 4 pg anti-IL-2 mAb clone 5355 (Sigma Aldrich, also designated MAB602 (Letourneau et al., 2010)), overnight at 4°C.
  • CD90.1 was stained with mAb OX-7 (ECACC), TCR Valpha2 with mAb B20.1 (Biolegend), TCR Vbeta5 with mAb MR9-4 (Becton Dickinson).
  • Example 1 Various modes of immunization result in a low-frequency of primed antigen-specific CD8 + T cells. These can be re-activated and expanded with the combination of ADAS and IL-2 with extended half-life.
  • C57BL/6 animals were injected on day 0 with a high amount (200 pg) of soluble, non-targeted OVA, or 2 pg 1 D3-OVA (targeted to B cells), or 2 pg muXCL1 -OVA (targeted to DC) by i.v. injection; in all cases, poly I: C (10 pg) was co-injected as a prototypical Th1 adjuvant which is a danger signal.
  • the initial immune response to OVA-derived peptide SIINFEKL SEQ ID No: 1
  • Some ADAS-treated animals were further injected i.p.
  • ADAS re-activation including 50 pg polyl:C as a prototypical Th1 adjuvant which is a danger signal
  • ADAS followed by application of IL-2ext for 3 consecutive days gave a frequency of 50-60% (Fig. 1 ).
  • This experiment determined the capacity of the combination of ADAS and IL-2ext to massively amplify a primary antigen-specific CD8 + T cell immune response, irrespective of the initial mode of immunization.
  • Example 2 For amplification of ADAS-re-activated antigen-specific CD8 + T cells IL-2ext is clearly superior to standard IL-2
  • C57BL/6 animals were primed on day 0 by i.v. injection with muXCL1 -OVA (2 pg) and poly l:C (10 pg) as a prototypical Th1 adjuvant which is a danger signal.
  • muXCL1 -OVA 2 pg
  • poly l:C 10 pg
  • ADAS i.v.
  • the proportion of SIINFEKL-specific CD8 + T cells was determined on day 10 in the spleen.
  • the IL-2 preparation MOPC-IL-2 with extended IL-2 half-life (IL-2ext)
  • MOPC-IL-2 was even clearly superior to 10-fold equimolar standard IL-2.
  • This experiment demonstrated the necessity to use IL-2 with extended half- life in order to achieve the desired massive expansion of pre-activated CD8 + T cells following the ADAS procedure.
  • Example 3 IL-2 complexed with an antibody blocking the CD25-binding epitope has comparable effects and adverse effects as various preparations of IL-2 with extended half-life (IL-2ext).
  • mice were immunized on day 0 with muXCL1 -OVA and poly I: C as an exemplary Th1 adjuvant which is a danger signal and received ADAS (0.5x10 6 SIINFEKL-loaded cells, 50 pg poly (l:C) i.v. on day 6. On days 7, 8, and 9, the mice were injected i.p.
  • hlL-2cx exhibited substantially stronger unwanted effects (expansion of T reg ) when compared to equimolar amounts of MOPC-IL-2, DP47GS-HC-IL-2, DP47GS-LC-IL-2, IL-2-HSA, and HSA-IL-2 (Fig. 3B).
  • Example 4 IL-2ext has to be applied within 5 days after ADAS
  • C57BL/6 animals were primed on day 0 with muXCL1 -OVA and an exemplary Th1 adjuvant which is a danger signal (poly (I: C)) and received ADAS (including poly (I: C) ) on day 6.
  • ADAS-treated mice were further injected with MOPC-IL-2 (IL-2ext) for 3 consecutive days, beginning either on day 7, or on days 8, 9, 10, or 11.
  • MOPC-IL-2 MOPC-IL-2
  • IL-2ext has to be applied within 5 days after ADAS to be effective in the expansion of ADAS re-activated CD8 + T cells.
  • Example 5 The combination of ADAS and IL-2ext re-activates and expands CD8 + T cells when applied 4 to 29 days after primary activation
  • ADAS antigen-specific T cells
  • IL-2ext C57BL/6 animals were primed on day 0 with muXCL1 -OVA (2 pg) and an exemplary Th1 adjuvant which is a danger signal, and re-activated with ADAS (including 50 pg poly (l:C)) on day 5 or day 6, 7, 8, or 9.
  • ADAS alone expanded the SIINFEKL-specific CD8 + T cell population (from around 3% on day 5, not shown) in each instance (Fig. 5A).
  • ADAS including 50 pg poly (l:C)
  • IL-2ext raising the frequency of SIINFEKL CD8 + T cells in all instances except on day 3.
  • IL-2ext reactivates primed T cells from day 4 up to day 30, when the CD8 + T cells have become memory T cells.
  • Example 6 The combination of ADAS and IL-2ext re-activates and expands memory T cells
  • Example 7 The combination of ADAS and IL-2ext massively expands recently in vitro activated and adoptively transferred CD8 + T cells
  • OT-I TCR-transgenic mice having a large percentage CD8 + T cells specific for the SIINFEKL-peptide (Hogquistet al. , 1994) were in vitro activated in the presence of SIINFEKL-peptide.
  • OT-I T cells as determined with antibodies to Thy1.1 and TCR Valpha2 and TCR Vbeta5 by flow cytometry
  • ADAS was applied (including poly (I: C)), some recipient animals were further injected with MOPC-IL-2 i.p. (4 pg) on days 6, 7, and 8.
  • Analysis of SIINFEKL-specific CD8 + T cells was on day 10 in the spleen.
  • the in vivo frequency of antigen-specific CD8 + T cells without amplification was around 1 % of all CD8 + T cells.
  • the frequency rose to around 10%.
  • ADAS and 3-day treatment with IL-2ext the frequency of OT-I T cells was 90% of all CD8 + T cells.
  • the frequency rose to around 25%. Additional 3-day treatment with IL-2ext further increased the frequency to around 90% (Fig. 7B).
  • CD8 + T cells will be amplified with ADAS and IL-2ext within the time frame of 4 to 29 days of in vitro activation.
  • OT-I T cells were re-activated by ADAS or ADAS+IL-2ext at a later time point, after they obtained memory status (day 42 after transfer).
  • CD8 + T cells will be amplified with ADAS and IL-2ext even after they have reached memory status.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical & Material Sciences (AREA)
  • Oncology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Communicable Diseases (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

La présente invention concerne une cellule présentatrice du CMH-I chargée de peptides et une IL-2 présentant une demi-vie prolongée pour une utilisation dans l'amplification d'une réponse immunitaire cytotoxique cellulaire et des méthodes associées.
PCT/EP2019/085667 2018-12-18 2019-12-17 Cellule présentatrice du cmh-i chargée de peptides et il-2 présentant une demi-vie prolongée pour amplifier une réponse immunitaire cytotoxique cellulaire Ceased WO2020127277A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP18213650.7 2018-12-18
EP18213650 2018-12-18

Publications (1)

Publication Number Publication Date
WO2020127277A1 true WO2020127277A1 (fr) 2020-06-25

Family

ID=65011724

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2019/085667 Ceased WO2020127277A1 (fr) 2018-12-18 2019-12-17 Cellule présentatrice du cmh-i chargée de peptides et il-2 présentant une demi-vie prolongée pour amplifier une réponse immunitaire cytotoxique cellulaire

Country Status (1)

Country Link
WO (1) WO2020127277A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009065561A2 (fr) 2007-11-20 2009-05-28 Bundesrepublik Deutschland Letztvertreten Durch Das Robert Koch-Institut Système pour une administration dans une cellule positive à xcr1 et ses utilisations
US20140044675A1 (en) 2012-08-10 2014-02-13 Roche Glycart Ag Interleukin-2 fusion proteins and uses thereof
WO2014201378A1 (fr) * 2013-06-13 2014-12-18 Massachusetts Institute Of Technology Traitement synergique de tumeur avec il-2 à pk prolongée et thérapie cellulaire adoptive
WO2015140172A2 (fr) 2014-03-17 2015-09-24 Bundesrepublik Deutschland Letztvertreten Durch Das Robert Koch-Institut Vertreten Durch Seinen Präsidenten Médicament destiné à être utilisé dans un procédé d'induction ou d'extension d'une réponse immunitaire cytotoxique cellulaire

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009065561A2 (fr) 2007-11-20 2009-05-28 Bundesrepublik Deutschland Letztvertreten Durch Das Robert Koch-Institut Système pour une administration dans une cellule positive à xcr1 et ses utilisations
US20140044675A1 (en) 2012-08-10 2014-02-13 Roche Glycart Ag Interleukin-2 fusion proteins and uses thereof
WO2014201378A1 (fr) * 2013-06-13 2014-12-18 Massachusetts Institute Of Technology Traitement synergique de tumeur avec il-2 à pk prolongée et thérapie cellulaire adoptive
WO2015140172A2 (fr) 2014-03-17 2015-09-24 Bundesrepublik Deutschland Letztvertreten Durch Das Robert Koch-Institut Vertreten Durch Seinen Präsidenten Médicament destiné à être utilisé dans un procédé d'induction ou d'extension d'une réponse immunitaire cytotoxique cellulaire
WO2015140175A1 (fr) 2014-03-17 2015-09-24 Bundesrepublik Deutschland Letztvertreten Durch Das Robert Koch-Institut Vertreten Durch Seinen Präsidenten Médicament destiné à être utilisé dans un procédé d'induction ou d'extension d'une réponse immunocytotoxique cellulaire

Non-Patent Citations (57)

* Cited by examiner, † Cited by third party
Title
"Fundamental Immunology", 2013
"Genbank", Database accession no. 3558
"GenBank", Database accession no. AAB62672.1
"UniProtKB", Database accession no. P60568
ARANDA F ET AL., ONCOIMMUNOLOGY, vol. 2, no. 12, 1 December 2013 (2013-12-01), pages e26621
BACHEM A ET AL., FRONT IMMUNOL., vol. 3, 18 July 2012 (2012-07-18), pages 214
BACHEM ET AL., FRONT IMMUNOL, vol. 3, 2012, pages 214
BACKER R ET AL., PROC NATL ACAD SCI USA., vol. 107, no. 1, 5 January 2010 (2010-01-05), pages 216 - 21
BINDER ET AL., CURR OPIN COLLOID INTERFACE SCI., vol. 31, 2017, pages 10 - 17
BOYMAN O ET AL., SCIENCE, vol. 311, no. 5769, 31 March 2006 (2006-03-31), pages 1924 - 7
BRAUNLEIN E ET AL., FRONT IMMUNOL., vol. 8, 30 November 2017 (2017-11-30), pages 1702
BROOKS NA ET AL., BIOCHIM BIOPHYS ACTA, vol. 1805, no. 1, January 2010 (2010-01-01), pages 25 - 34
CAMINSCHI ET AL., BLOOD, vol. 112, 2008, pages 3264 - 3273
CARROLL EC ET AL., IMMUNITY, vol. 44, no. 3, 15 March 2016 (2016-03-15), pages 597 - 608
CARY F OPEL ET AL: "Combined treatment using adoptive cell therapy, extended pharmacokinetic IL-2, and tumor-specific antibodies leads to cures of established B16F10 tumors and extended in vivo T cell survival", JOURNAL FOR IMMUNOTHERAPY OF CANCER, BIOMED CENTRAL LTD, LONDON, UK, vol. 1, no. Suppl 1, 7 November 2013 (2013-11-07), pages P26, XP021167220, ISSN: 2051-1426, DOI: 10.1186/2051-1426-1-S1-P26 *
COFFMAN RL ET AL., IMMUNITY, vol. 33, no. 4, 29 October 2010 (2010-10-29), pages 492 - 503
DONOHUE JH ET AL., J IMMUNOL., vol. 130, no. 5, May 1983 (1983-05-01), pages 2203 - 8
DUNNE A ET AL., MUCOSAL IMMUNOL., vol. 8, no. 3, May 2015 (2015-05-01), pages 607 - 17
FARBER DL ET AL., NAT REV IMMUNOL., vol. 14, no. 1, January 2014 (2014-01-01), pages 24 - 35
GURKA S ET AL., FRONT IMMUNOL., vol. 6, 4 February 2015 (2015-02-04), pages 35
HARTUNG E ET AL., J IMMUNOL., vol. 194, no. 3, 1 February 2015 (2015-02-01), pages 1069 - 79
HENSLER M ET AL., ONCOIMMUNOLOGY, vol. 5, no. 4, 10 December 2015 (2015-12-10), pages e1102827
HOGQUIST KA ET AL., CELL, vol. 76, no. 1, 14 January 1994 (1994-01-14), pages 17 - 27
HULEATT JW ET AL., VACCINE, vol. 25, no. 4, 8 January 2007 (2007-01-08), pages 763 - 75
IMANISHI T ET AL., J IMMUNOL., vol. 178, no. 11, 1 June 2007 (2007-06-01), pages 6715 - 9
KASTENMULLER W ET AL., NAT REV IMMUNOL., vol. 14, no. 10, October 2014 (2014-10-01), pages 705 - 11
KIM WS ET AL., ONCOTARGET., vol. 7, no. 18, 3 May 2016 (2016-05-03), pages 24962 - 82
KRIEG C ET AL., PROC NATL ACAD SCI U S A., vol. 107, no. 26, 29 June 2010 (2010-06-29), pages 11906 - 11
KROP I ET AL., EUR J IMMUNOL., vol. 26, no. 1, January 1996 (1996-01-01), pages 238 - 42
LETOURNEAU S ET AL., PROC NATL ACAD SCI USA., vol. 107, no. 5, 2 February 2010 (2010-02-02), pages 2171 - 6
MERAD M ET AL., ANNU REV IMMUNOL., vol. 31, 2013, pages 563 - 604
MITTRUCKER HW ET AL., ARCH IMMUNOL THER EXP (WARSZ, vol. 62, no. 6, December 2014 (2014-12-01), pages 449 - 58
NETEA MG ET AL., ANTIMICROB AGENTS CHEMOTHER, vol. 49, no. 10, October 2005 (2005-10-01), pages 3991 - 6
PASUT G., POLYMERS, vol. 6, no. 1, 2014, pages 160 - 178
POTTER ET AL., J NATL CANCER INST, vol. 26, 1961, pages 1109 - 1137
POTTER M ET AL., J NATL CANCER INST., vol. 26, May 1961 (1961-05-01), pages 1109 - 37
ROJAS G ET AL., IMMUNOBIOLOGY, vol. 218, no. 1, January 2013 (2013-01-01), pages 105 - 13
ROSENBERG SA ET AL., SCIENCE, vol. 348, no. 6230, 3 April 2015 (2015-04-03), pages 62 - 8
S. LETOURNEAU ET AL: "IL-2/anti-IL-2 antibody complexes show strong biological activity by avoiding interaction with IL-2 receptor ? subunit CD25", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 107, no. 5, 19 January 2010 (2010-01-19), pages 2171 - 2176, XP055182715, ISSN: 0027-8424, DOI: 10.1073/pnas.0909384107 *
SANDER B ET AL., J IMMUNOL METHODS, vol. 166, no. 2, 3 December 1993 (1993-12-03), pages 201 - 14
SCHELLENBERGER V ET AL., NAT BIOTECHNOL., vol. 27, no. 12, December 2009 (2009-12-01), pages 1186 - 90
SCHWENDENER RA ET AL., THER ADV VACCINES, vol. 2, no. 6, November 2014 (2014-11-01), pages 159 - 82
SILVA-GOMES S ET AL.: "Ecyclopedia of Inflammatory Diseases", 2014, SPRINGER, pages: 1 - 16
SKOBERNE M ET AL., TRENDS MOL MED., vol. 10, no. 6, June 2004 (2004-06-01), pages 251 - 7
SPEISER DE ET AL., SEMIN IMMUNOL., vol. 22, no. 3, June 2010 (2010-06-01), pages 144 - 54
SPROKHOLT JK ET AL., J IMMUNOL., vol. 198, no. 12, 15 June 2017 (2017-06-15), pages 4764 - 4771
TACKEN PJ ET AL., SEMIN IMMUNOL., vol. 23, no. 1, February 2011 (2011-02-01), pages 12 - 20
TAKAI Y ET AL., CANCER SCI., vol. 94, no. 8, August 2003 (2003-08-01), pages 655 - 67
TOUSSI DN ET AL., VACCINES (BASEL, vol. 2, no. 2, 25 April 2014 (2014-04-25), pages 323 - 53
TROVATO M ET AL., WORLD J VIROL., vol. 4, no. 3, 12 August 2015 (2015-08-12), pages 156 - 68
VAN MONTFOORT N ET AL., FRONT IMMUNOL., vol. 5, 23 April 2014 (2014-04-23), pages 182
VERONESE FM ET AL., BIODRUGS, vol. 22, no. 5, 2008, pages 315 - 29
WALSENG E ET AL., SCIENTIFIC REPORTS, vol. 7, 2017, pages 10713
WANG X ET AL., CHEM SOC REV., vol. 42, no. 12, 21 June 2013 (2013-06-21), pages 4859 - 66
WANG Y ET AL., J IMMUNOL., vol. 169, no. 5, 1 September 2002 (2002-09-01), pages 2422 - 9
WU C ET AL., PROC NATL ACAD SCI USA., vol. 108, no. 22, 31 May 2011 (2011-05-31), pages 9178 - 83
YAMADA A ET AL., CANCER SCI., vol. 104, no. 1, January 2013 (2013-01-01), pages 15 - 21

Similar Documents

Publication Publication Date Title
US20240082373A1 (en) Compositions for chimeric antigen receptor t cell therapy and uses thereof
US20230119334A1 (en) Antigen receptors and uses thereof
US10688165B2 (en) Medicament for use in a method of inducing or extending a cellular cytotoxic immune response
RS60089B1 (sr) Novi kompleks koji sadrži peptid koji penetrira u ćeliju, teret i tlr peptidni agonist za tretman kolorektalnog kancera
JP2025069162A (ja) 免疫調節融合タンパク質－金属水酸化物錯体およびその方法
WO2020191305A2 (fr) Utilisations d'amphiphiles en thérapie cellulaire immunitaire et compositions associées
US12433954B2 (en) Methods of activating anti-CD19 chimeric antigen receptor (CAR) T cells using amphiphilic ligand conjugates comprising CAR-targeting protein sequence motifs
Pappalardo et al. Characterization of a nanovaccine platform based on an α1, 2-mannobiose derivative shows species-non-specific targeting to human, bovine, mouse, and teleost fish dendritic cells
WO2021221783A1 (fr) Procédés d'identification de ligands de ciblage de récepteur antigénique chimérique et leurs utilisations
WO2020127277A1 (fr) Cellule présentatrice du cmh-i chargée de peptides et il-2 présentant une demi-vie prolongée pour amplifier une réponse immunitaire cytotoxique cellulaire
US20240100161A1 (en) Cytosolic delivery
JP2020509783A (ja) 抗原受容体及びその使用
US20230210968A1 (en) Ribonucleoprotein approach to boost the sting signaling for cancer immunotherapy
Al-Khami et al. Adoptive immunotherapy of cancer: Gene transfer of T cell specificity
US20250302970A1 (en) Uses of amphiphiles in immune cell therapy and compositions therefor
RU2819805C2 (ru) Композиции для т-клеточной терапии с химерным антигенным рецептором и их применения
Moseman Delivery of Plasmid DNA Vaccines With Checkpoint Blockade or Lipid Nanoparticles to Improve Anti-Tumor Efficacy
Sittplangkoon mRNA vaccine encoding neoantigen for cancer immunotherapy using mouse melanoma as a model
WO2023288056A2 (fr) Compositions et procédés pour la multiplication de populations de lymphocytes t restreints par hla-e
Shen Self-Assembled Antibody Nanorings as Prosthetic Antigen Receptors for Redirecting T Cells against Tumor Cells and as Platform for Delivery of Vaccine Adjuvants for Cancer Immunotherapy
Xiao Dendritic cell-specific vaccine utilizing antibody-mimetic ligand and lentivector system
HK40018015A (en) Antigen receptors and uses thereof
Moran Characterization of dendritic cells transduced with Venezuelan equine encephalitis virus replicon particles as therapeutic cancer vaccines
HK1259455B (en) Antigen receptors and uses thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19827691

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19827691

Country of ref document: EP

Kind code of ref document: A1